Antioxidant activity of Myxobacteria isolated from soil.pdf

NGUYEN TAT THANH UNIVERSITY --- oOo---SUMMARY REPORT ON STUDENT PROGRAM SCIENTIFIC RESEARCH 2020 Topic’s name: ANTIOXIDANT ACTIVITY OF MYXOBACTERIA ISOLATED FROM SOIL Topic’s code: Topi

NGUYEN TAT THANH UNIVERSITY

-

oOo -SUMMARY REPORT ON STUDENT PROGRAM

SCIENTIFIC RESEARCH 2020

Topic’s name:

ANTIOXIDANT ACTIVITY OF MYXOBACTERIA

ISOLATED FROM SOIL

Topic’s code:

Topic manager: NGUYEN THI KIM HONG

Scientific instructor: M.Sc Pharm NGUYEN THI NGOC YEN

Industry: Pharmacology

The members participate:

Ho Chi Minh city, 10/2020

LIST OF ACRONYMS iv

LIST OF TABLES V LIST OF FIGURES vi

ABSTRACT vii

INTRODUCTION 1

CHAPTER 1 LITERATURE REVIEW 3

1.1 The SOIL MICROBIOTA ANDPOTENTIAL SECONDARY METABOLITES 3

1.1.1 Role inorganisms 3

1.1.2 Role inecosystem 3

1.1.3 Role inmedical use 4

1.2 THE MYXOBACTERIA 4

1.2.1 The ecologyof the myxobacteria 5

1.2.2 Genome 5

1.2.3 Enzymatic activity 6

1.2.4 Phylogeny of Myxobacteria 8

1.2.5 Morphological facts seem to explain the special traits of the myxobacteria 10

1.3 THE DISCOVERY OF BIO ACTIVE METABOLITES 15

1.4 ISOLATION AND IDENTIFICATION 16

1,4.1 Isolation ofmyxobacteria 16

1.4.2 Purification of isolates 18

1.4.3 Identify myxobacteria strains 18

1.5 ANTIOXIDANTION ACTIVITY AND MICROBIAL SOURCE 19

1.5.1 Antioxidant agents and myxobacteria 19

1.5.2 Folin-Ciocalteu assay for polyphenols total 20

1

CHAPTER 2 MATERIALS AND RESEARCH METHODS 22

2.1 SUBJECTS AND MATERIALS 22

2,1.1 Subjects 22

2,1.2 Instruments and equipments 22

2.1.3 Media and chemicals 23

2.2 RESEARCH METHODS 24

2.2.1 Samples collection and storage conditions 24

2.2.2 Treatment of soil samples 24

2.2.3 Isolation of myxobacterial strains 25

2,2.4 Purification methods 26

2.2.5 Storage of isolates 27

2,2,6 Identify the Myxobacteria strains 27

2,2.7 Extraction of myxobacterial crude extract 29

2.2.8 Screeningantioxidant activity 31

CHAPTER 3 RESULTS AND DISCUSSION 34

3.1 Collection of samples 34

3.2 Isolation and identification 35

3.2.1 Isolation 35

3.2.2 Identification 36

3.2.3 Discussion 43

3.3 Screeningantioxidant activity 45

3.3.1 Folin-Ciocalteumethod for total polyphenols 45

3.3.2 ABTS radicalcation scavenging activity 48

3.3.3 Discussion 51

CHAPTER 4 CONCLUSION AND SUGGESTION 53

4.1 CONCLUSION 53

iii

CFU Central Processing Unit

LB broth Luria Bertani Broth

P-medium Peptone medium

wcx Wateragarwith cycloheximide 100 pg/ml

GAE Gallic acid equivalent

TEAC Trolox equivalentantioxidant capacity

ANOVA Analysis of variance

Table 1.1 Overview of myxobacterial strains found in soil 9

Table 1.2 A listof published bioactive compounds 16

Table 2.1 List of laboratory instruments used in this research 22

Table 2.2 Some chemicals and reagents areused throughout theprocess 23

Table 3.1 Statistical table of soil samples collected in Vietnam 34

Table 3.2 The image of morphological Myxobacteria 36

Table 3.3 The number of identified strainsfrom different provinces 38

Table 3.4 Myxobacterial strains isolatedby differentmethods 39

Table 3.5 Identitification of new isolates based on 16SrDNA analysis 40

Table 3.6 Data for establishing gallic acid calibration curve 45

Table 3.7 Data of absorbance and concentration of each sample 46

Table 3.8 ANOVA analysis of polyphenol content data of samples 48

Table 3.9 Data for establishing Trolox calibration graph 48

Table 3.10 The Trolox equivalent concentration of extracts from p and VY3 media 49 Table 3.11 ANOVA analysis of polyphenol content data of samples 51

V

Figure 1.1 Group and solitary-based predation by Myxococcus xanthus 1

Figure 1.2 Stereophotomicrographs ofByssovorax cruenta on filter paper 8

Figure 1.3 Taxonomy of Myxococcales 9

Figure 1.4 Thepredatory life cycle ofMyxococcus xanthus 10

Figure 1.5 Stereophotomicrographs of swarm colony surface Chondromyces 11

Figure 1.6 Morphology of twomyxobacteria suborders 12

Figure 1.7 The variety of myxobacterial fruiting bodies 14

Figure 1.8 Wild rabbit dung 17

Figure 1.9 The reaction mechanism of 2,2’-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) with antioxidant 20

Figure 2.1 The study procedure 24

Figure 2.2 Illustrationof three isolation methods 26

Figure 2.3 The myxobacterial purification 27

Figure 2.4 Gram staining procedure 28

Figure 2.5 Decomposing filter paper 28

Figure 2.6 Mega X and phylogenetic tree 29

Figure 2.7 The procedure of crude extract preparation 31

Figure 2.8 The mechanism ofFolin-Ciocalteu method 32

Figure 3.1 Apperances of fruiting bodies from isolation 35

Figure 3.2 The gram-stained images of vegetative cells 37

Figure 3.3 The number of 43 myxobacterial strains isolated 39

Figure 3.4 Myxobacterial strains isolated by different methods 39

Figure 3.5 Gallicacid calibration curve in the Folin-Ciocalteu method 45

Figure 3.6 The sample concentration expressed as gallic acidequivalents 47

Figure 3.7 Trolox calibration curve in ABTS assay 49

Figure 3.8 The sample concentration expressed as Trolox equivalents 50

DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUIREMENTS

FOR THE PHARMACIST - ACADEMIC YEAR 20 - 20 ANTIOXIDANT ACTIVITY OF MYXOBACTERIA

ISOLATED FROM SOIL

Nguyen Thi Kim Hong

Supervisor: M.Sc Pharm Nguyen Thi Ngoc Yen Introduction: The objectives of thesis is to explore new strains capable of producing secondary metabolites with antioxidant activity that opened promising treatment direction

on cancer disease in the future.

Materials and methods: Myxobacteria were isolated using three conventional methods (WCX with E coli bait, filter paper, and rabbit dung) Myxobacterial with morphological characteristics and 16S rRNA gene sequence analysis were identified Antioxidant activities were evaluated by (i) The Folin-Ciocalteu method for total phenol content and (ii) The ABTS

radical cation scavenging activity

Results: From 110 soil samples collected at 19 cities and provinces, 43 identified strains /68 pure isolates with high similarity (from 91.1-100%) were assigned in six genera, including

Archangium, Angiococcus, Chondromyces, Corallococcus, Cystobacter, and Myxococcus.

The total contents of polyphenols are ranged from 7.522 to 67.864 pg GAE equivalents/ml and 0.250 - 89.776 pg GAE/ml for extracts from P-medium and VY3, respectively Extract

of VL 2.5 gave the highest polyphenol total content in both culture media.

In ABTS method, the Trolox concentration ranges from 2.508 ±0.199 to 13.428 ±0.160 pg TEAC/ml, in which, the HG 2.25 extract exhibits the highest activity (equivalent 13.428 ± 0.160 pgTEAC/ml) InVY3 medium, the Trolox values ranges from 2.031 ±0.138 to 14.010

± 0.142 pg TEAC/ml with the highest activity is GL 4.3 (equivalent 14.010 ± 0.142 pg TEAC/ml).

Conclusion: Most of the myxobacterial strains exhibited antioxidant activity that has

different concentrations and depends on the culture media The results are statistically significant differences in the Folin-Ciocalteu method and ABTS assay So, each strain must choose a suitable medium for fermentation The HG 2.25, HCM 8.1, VL 2.5, GL 4.3, TH 8.2 were the potential strains with the best antioxidant activity to proceed to the next active screening steps, e.g., antitumor activity.

vii

In recent years, there has been a great deal of research toward the free radical fields Various endogenous systems generate free radicals in our body, and a close relationship was demonstrated between an increase in free radicals and the risk ofexposure to pathological states The high concentrations of free radicals promote oxidation, damage cellular structures, or biologically relevant molecules such asDNA, proteins, carbohydrates, and lipids Free radicals attack essential macromolecules leading to cell damage and degenerative diseases, cardiovasculardisease, atherosclerosis, hypertension, diabetes, arthritis, weakened immune system, and the phenomenon of premature aging Therefore, a balance between free radicals and antioxidants is necessary for proper physiological function If free radicals overwhelmthe body's ability of regulation,the supplement of antioxidants is essential

to control them

The research on antioxidants is increasingly interested in reducing free radicals andpreventing diseases in the most effective way The use of a source of antioxidants from microorganisms is dominant because of their broad distribution in nature, easy cultivation on a large scale, low-costcultural media, andrapid growth [2], [14].Myxobacteria is also one of the most promising strains since they were functionallycharacterized to assess their ability to produce antioxidant, antimicrobial, andanticancermetabolites Most compounds frommyxobacteria were not found in otherorganisms and are unique to the bacterium [5], [20]

According to our research, up to now, research on the biological activities of themyxobacterial extracts is still limited in Vietnam Therefore, this study is conducted

to evaluate the antioxidant activity ofcrude extract from myxobacteria with the aim

offindingmore sources of the natural microbial system to protect human health The screening of active substances with antioxidant activity from myxobacteria strains is

a beginning step for the detection of strains capable of producing anticancer substances So the thesis named "Antioxidant activityofmyxobacteria isolated fromthe soil" was performed to satisfy four objectives:

- Isolation ofmyxobacteria strainswith three methods: (1) Isolation using wild rabbit dung, (2) Isolation using E coll baiting on wcx medium, and (3)Isolation of cellulolytic myxobacteria with ST21 medium (using filter paper);

- Purification and storage ofmyxobacteria strains;

- Identification using morphological characteristics, gram staining, or 16S rRNA analysis;

- Preparation of crude extracts from purified strains and evaluation of theantioxidant activity using the ABTS radical cation scavenging activity andFolin -Ciocalteu method

2 Nguyen Thi Kim Hong

CHAPTER 1 LITERATURE REVIEW

1.1 THE SOIL MICROBIOTA AND POTENTIAL SECONDARY METABOLITES

Microorganisms are organisms that are small enough to be microscopic In otherwords, they require a microscope to be seen Examples of types ofmicroorganismsinclude bacteria, protozoa, viruses, and fungi In this lesson, we're going to take alook at howtheyimpact large organisms andecosystems, bothfortheir good and bad

1.1.1 Role in organisms

Inside organisms like humans, microorganisms canbe wonderfully useful, orterriblydestructive Most people know that bacteria and viruses can make you sick, and thatsome can even be deadly; For example, we all have bacteria and viruses in ourintestines whichhelp to digest food and make the environment inhospitable for more dangerous microorganisms In fact, after being treated with antibiotics, you might find you get sick notlong after becausethe antibioticsmay have killed useful bacteria

in your intestines By killing off safe bacteria, the dangerous invaders have nocompetitionandcanthen spread more easily Having so many microorganisms inside your body also serves as a training ground for your immune system, making itstronger There is even some evidence that microorganisms absorb toxins and help reduce feelings ofstress [3]

1.1.2 Role in ecosystem

Microorganisms are found everywhere in the environment and play a leading role incountless natural processes Among other things, they operate the basic cycles thatare necessary for the plants' supply of nutrients via the reaction oforganic matter in soil At these processes, greenhouse gases arereleased to theatmosphere at the sametime, so microorganisms also play a key role in relation to climate and climate change

Animals are only one small part of an ecosystem Foran ecosystem towork, it has tohave many organisms that all work together in a continuous cycle Microorganismsform part of that cycle, and because oftheir huge numbers, the part they play is an important one Microorganisms have several vital roles in ecosystems:decomposition, oxygen production, evolution, and symbiotic relationships

1 Generate oxygen in atmosphere: Almost all ofthe oxygen produced in earth today occurs by bacteria in prehistoric period Most of the oxygen producers arecyanobacteria (blue green algae)in ocean.

2 Recycle nutrition stored in organic matters to inorganic form: Microbial decomposition of organic matters releases the minerals such as N, p, K and theseminerals are bound up and made available for producers (green plants) to use Without this recycling primary productivity of ecosystem would stop In soil, fungi are the mostimportant decomposers ofplant materials and are followed by bacteria

3 Fix atmospheric nitrogen into useable form: The only organism capable of fixingatmospheric nitrogen into useable form are bacteria These nitrogen fixing bacteriaare Rhizobium, Cyanobacteria, e.g

4 Microorganisms give plant roots excess to nutrition in soil: Plant root createszone of nutrition depletion around their surface by using available nutrition

5 Allows herbivore animals to get nutrition from poor quality food:

1.1.3 Role in medical use

Inaddition toexercising their functionin theenvironment, microorganisms also have

a great potential for use Some bacteria and fungi, so-called biocontrol organisms, can inhibit the growth of harmful microorganisms, technical enzymes and enzymesfor food production can be isolated from bacteria, yeasts and fungi, which are alsomajor suppliers of pharmaceuticals such as e.g antibiotics.In soil ecosystems, microorganisms produce diverse secondary metabolites such as antibiotics, antifungals and siderophores that mediate communication, competition andinteractions with other organisms and the environment Most known antibiotics arederived from a few culturable microbial taxa, and the biosynthetic potential ofthevast majority of bacteria in soil has rarely been investigated [3]

Myxobacteria are rod-shaped Gram-negative eubacteria with relatively large measuring 0.6-1.2 pm inwidth and3-15 pm in length, which move by gliding on thesurface They are very widespread and ubiquitous organisms It has been isolated insoil, bank and trees, decaying plant material, and special habitats like bogs, rottingwood, or from marine sources and freshwater environments Myxobacteria have a

4 Nguyen Thi Kim Hong

complex life cycle that includes vegetative swarming, predation, and fruiting body formation in starvation conditions [21].

1.2.1 The ecology of the myxobacteria

The myxobacteria are found nearly everywhere It very common and seem to be aubiquitous group of organisms that can inhabit very diverse habitats The typical habitat of myxobacteria is soil All known of them are strictlyaerobic and live in thetopmost layers of the soil Besides, they colonize decaying plant material, includingrotting wood and bark from dead and living trees, decomposing lichens and insects,and dung, especially certainherbivorous mammals, such as wild rabbits,deer, sheep,and goats Rabbit dung is a classical source for the isolation of myxobacteria

The aquatic habitats appear not to be very typical for myxobacteria, but the strainslike Enhygromyxa, Plesiocystis, Pseudenhygromyxa, and Haliangium was isolated from marine sources Sometimes, myxobacteria have also been found in special habitats such as in peat bogs (Myxococcus, Corallococcus, and Polyangium sp.) at

pH 3.2-4.8

MostMyxobacteria are strictly aerobic mesophiles with a narrow pH rangein culture

is usually 6.8-7.8 (used pH 7 for isolation) However, there are still some strainsisolated in alkaline or acidophilic The salt tolerance of myxobacteria appears to berelatively low However, somemyxobacteria tolerate salt betterthanothers, so added

of0.5% NaCl to isolation agar stimulates the development (Using wcx agar with streaks of livingE coli for isolatedthe myxobacteria such as M virescens). Almost all Myxobacteria grow well at 30°C, although their temperature range is much broader In the laboratory, the useful temperature range for cultivating myxobacteria

is 28-34°C (approximately the room temperature), with generation times in 4-14 hours The vegetative cells are somewhat sensitive to desiccation opposites; themyxospores are completely desiccation resistant Therefore, attention should bepaid

to the conditions for storingstrains [21], [23], [26]

1.2.2 Genome

Myxobacteria possess giant chromosomes belonging to the largest genome of allknown bacteria A genome size rangingfrom 5690 to 12,727 kbp and a DNA with aG+C contentof66-72 mol%was noted Most myxobacteria arecomposed ofa singlecircular chromosome and no extra-chromosomal plasmids Until 1980, when the first

endogenous plasmid was identified in Myxococcus fulvus, it was not found in M xanthus. Amongthemyxobacteria, M xanthusis studiedmost genetically; it contains

a much higher G+C proportion than doesE coll which has 67.5 mol% G+C The G+C content of strains will be ranged from 70.0-73.4% (the strain - Corallococcus

coralloides had 73.39 mol% G+C) [23], [26], [28],

1,2.3 Enzymatic activity

All myxobacteria are characterized by their ability to degrade biological macromolecules They are divided into two groups depending on their ability toutilize inorganic nitrogen compounds: (1) The cellulolytic myxobacteria; (2) The bacteriolytic Myxobacteria In the environment, each group has an important role Bacteriolytic myxobacteria are micro predators that impact the carbon flow andcomposition of microbial communities, while cellulolytic myxobacteria aresignificant global carbon cycle as degradersof plant biomass

Proteolytic enzymes are produced both by the cellulolytic myxobacteria (e.g.,members of the genus Sorangium — Sorangium cellulosuni), as well as by thepredatory ones (e.g., belonging to the genus Myxococcus - Myxococcus xanthus)

[15], [5], [26]

a The bacteriolytic myxobacteria

Bacteriolytic myxobacteria that lyse whole cells of other living microorganismsrepresent the majority of the myxobacterial species Nitrogen source is primarily supplied by enzymatic degradation ofproteins as oligopeptides orsingle aminoacids which containing growth substrates (peptones) Because myxobacteria are capable oflysing the living cells, so they are called predators

The production of proteolytic enzymes (e.g., lipase, nuclease, polysaccharides vàprotease) and cell wall degrading to be involved in the lysis ofprey microbes Like protease has three possible functions: (i) proteases may supply amino acids to themyxobacteria by hydrolyzing soil proteins derived from plant, animal, and soil microorganisms, (ii) after the activity of cell wall lytic enzymes, a protease may disrupt the cell membrane of the eubacterium, releasing its intracellular content accessible to the surrounding environment, and (iii) the ultimate lysis ofthe prey islikely to involveproteases Thiscouldbe seen in strain Myxococcus.

6 Nguyen Thi Kim Hong

Figure 1.1 showed: (a) Time course of solitary predation by myxobacteria FP-1 oncyanobacteria cells The cyanobacterial species can be observed as a chain of large, spherical cells The arrow points to a rod-shaped myxobacter cell in the process oflysing two cyanobacteria cells (b)Time course ofM xanthus invading and lysing acolony of Escherichia coli prey bacteria (c)These images show the three majormorphological traits of group-mediated predation: colony invasion (top), rippling wave structures (middle), and fruiting bodies (bottom) [1].

Figure 1.1 Group and solitary-based predation byMyxococctts xanthus

b The cellulolytic myxobacteria

The cellulolytic myxobacteria that efficiently decompose cellulose (cellulose degraders, suborder Sorangiineae) can utilize inorganic nitrogen compounds

The members ofthe genus Sorangium represent for cellulolytic activity They have the protuberant structure on the surface that is responsible for cellulose degradation, and cellulose materials destroyed were limited to the region of a cellular swarm Cellulolytic enzymes are produced in Sorangium, which are arranged into a complex

of 1000 -2000 kDa, included cellulase and xylanase activities

Example in figure 1.2: Isolation image ofByssovorax cruenta strain belonging to suborder Sorangiineae - family Polyangiaceae on filter paper [11].

Figure 1.2 Stereophotomicrographs ofByssovorax cruenta on filter paper

8 Nguyen Thi Kim Hong

myxobacteria closely related to sulfate-reducing bacteria and Bdellovibrio species,which are also predators of bacteria [23], [26].

Snrunjyincjc

CyilobiKtcrincac

Sormpum fanciu IVIyanfium Actkwhacca Ihpbungiutn IlywunwAt ChiirMliinntc*

kMCTTIKydl*

ịptuudicynídKtac

Mills.

Figure 1.3 Taxonomy of Myxococcales

Up to now, more than a hundred publications have appeared that deal with theoccurrence regions reported in table 1.1 [4]

Table 1.1 Overviewof myxobacterial strains found in soil

Myxococcus fulvus, stipitatus, virescens, xanthus

Corallococcus coralloides, exiguus

Archangium gephyra, serpens

Sorangi urn cellulosum

sorediatum, spumosum, thaxteri, vitellinum

Chondromyces apiculatus, catemdatus, crocatus, lanuginosus,

pediculatus Nannocystis exedens

1.2.5 Morphological facts seem to explain the special traits of the myxobacteria

The myxobacteria are ubiquitous organisms which are famous for three capabilities: (1) They move by gliding, andtheir colonies are therefore thin, film-like swarms thatspread gradually over the culture plate; (2) They have a sophisticated intercellularcommunication system and a highly developed social life; (3) They show aremarkablemorphogenetic potential,which is expressedon two levels [26], [21]

Figure 1.4 The predatory life cycleofMyxococctts xanthus

1,2.5.1 Morphology of myxobacterial strains

Myxobacteria occur in at least two morphological types: (1) Cylindrical rods withrounded ends; and (2) Slender flexible rods with more orless tapering ends,

a Colony

“Swarming behavior" of the myxobacteria is a multicellular spreading into an unoccupied area, which is a characteristical trait of myxobacterial colonies Thisspreading is calledswarming, the colony is called "swarm" that is seen by the increase

ofthe colony diameter and is mainly due to the motility of single cells, to a lesser

10 Nguyen Thi Kim Hong

extent to cellmultiplication The spreadingrate is a measure of thegliding activity ofthe cells The motility of myxobacteria is determined by the original cell density,nutrient concentrations, and temperature.

Figure 1.5 Stereophotomicrographs of swarm colony surface Chondromyces

The swarming behavior of myxobacteria was different between strains In thesuborderCystobacterineae, swarms of vegetativecellsarefatandthin layered usually remain on the surface ofthe agar and tend to make strikingpatterns Their colonies absorb the diazo dyeCongo redand alsoform ridges ofcells known as ripples, whichmove processively in waves In contrast, swarms ofthe suborder Sorangiineae areoften embedded in theagar, sometimes formingpits intheagarsurface, andthey may not produce pronounced swarming patterns on surfaces However, their colonies do not adsorb Congo red or move in ripples [23], [28], [4]

b Vegetative cells

Myxobacteria cells are rod-shaped relatively large; depending on the species, theyare 0.6-1.2 pm inwidthand3-15 pm in length Vegetative cells occur in two differenttypes:

Cell type I: Suborder Cystobacterinae have long, a slender vegetative cell withtapered ends, usually tipped flexible rods up to 1 pmin diameter and up to 20 pm in

length and that change their shape considerably during myxospore formation,(members of families Myxococcaceae, Archangiaceae, and Cystobacteraceae).

Celltype II: Suborder Sorangiineae have a short vegetative cell withrigid cylindrical rods with rounded ends/ blunt ends up to 1 pm wideand up to 10 pm long thatchangetheir shape little upon myxospore formation (a memberof the family Sorangiaceae, also called Polyangiaceae) [4], [23]

c Myxospores

The myxospores are generated from cellular morphogenesis, which the vegetative cells shorten and fatten They become rod-shaped or spherical, are surrounded by acapsule in some cases

Figure 1.6 Morphology of two myxobacteria subordersVegetative cells of (a) suborder Cystobacterineae, (b) suborderSorangiineae;Myxospores of (c) Myxococcus fulvusand (d) Cystobacter ferrugineus [22].The myxospores show capable an increased resistance against ƯV - irradiation, mechanical stress, elevated temperatures (58 to 60°C in aqueous suspensions), andcompletely desiccation resistant That can help the myxospores survive unfavorable

12 Nguyen Thi Kim Hong

environmental conditions and can be stored in the laboratory in the dry conditionformany years without losing their viability.

The morphology of myxosporeswas different between threestrains of myxobacteria

In suborder Cystobacterineae, myxospores are much shorter and rounder and tend to have capsules that differ the suborder Sorangiineae, tend to be similar in shape tovegetative cells, and do not have visible capsules Nevertheless, that are alwaysgrouped into sporangioles have thick-walled casings for groups of myxospores The Nannocystineae are closely related to the Sorangiineae, and they may produce sporangioles or spores, butthat species do not produce fruitingbodies [20], [24],

d Fruiting bodies

The morphology of myxobacteria varies depending on environmental conditionsthroughout their characteristic life cycle Under stress conditions, vegetative cells aggregate to form multicellular structures known as fruiting bodies Within thesestructures, cells continue to change to form myxospores (contains 100.000 myxospores), which can endure stressful circumstances (e.g., low nutrientlevels) forlong periods When conditions improve, the myxospores germinate into vegetative cells

The color offruiting bodies may be white, brown, or bright yellow, orange, red or lilac, and size varies between 10 and 100 pm They have two morphologies: stalked and non-stalked fruiting bodies It can be seen with the naked eye and are easy torecognize under a dissecting microscope The fruitingbody has very diverse shapesand makes the difference betweenstrains In three suborders, the Nannocystineae do not produce fruiting bodies, although they may produce sporangioles orspores [4] For example in Figure 1.6: (A) Stigmatella erecta on the surface ofminimal casitone agar;(B) Cystobacter sp onVY/2 agar; (C) Chondromyces apiculatus, top view onrabbit dung bait; (D) Melittangium boletus, the early stage of development, ona piece

of plantmaterial adhering to rabbit dung; (E) Sorangium cellulosum onfilteredyeastagar(cVY/2); and (F) Myxococcus sp on casitone agar [7]

Figure 1.7 The varietyof myxobacterial fruiting bodies1.2.5.2 Glidingmotility

The myxobacterial cells move by gliding or creeping when in contact with a suitable substrate, e.g., an agar or a glass surface However, when suspended in liquid, they are non-motile The glidingspeed is 10-60 pm/min

To swam toward new nutrient sources, myxobacteria depend on two genetically and physiologically distinct motility systems that is (1) Adventure motility (A) (individual) and/or (2) Social motility (S)

Myxococcus xanthus was well researched about that M xanthus has two separate systems for gliding, adventurous (A)- and social (S)- motility S-motility, also called the social system, is responsible for the movement ofcells in groups, whereas A- motility is required for the movement of individual cells A large number of geneproducts play a role in A- and S-motility The motility that provides the twitching motility force for S-motility is generated by the extension and retraction oftype IV

14 Nguyen Thi Kim Hong

pili, which are usually present onlyat one pole ofthe cell at onetime and are polarlylocalized The S-motility and A-motility systems are synergistic, as colony spreading

by wild-type cells is faster than the sum of those of individual cells (A+S- andA-S+) In the absence of one orboth motility systems, the aggregation, fruiting bodyformation, and rippling are defective, indicating that motility is required for these social behaviors [26], [4]

The primary producers of secondary metabolites are members ofActinomyces (ca

8000 compounds characterized), the genus Bacillus (1400 metabolites), as well as

Pseudomonas (400 metabolites) Nevertheless, aside from fungi and actinomycetes,myxobacteria are also critical sources for natural microbial products and are knownfortheirexceptionalability to produce structurally diverse secondary metabolites So, over the last decade, theyhave emerged as a promising alternative source of bioactivemolecules The difficulty in cultivating myxobacterial strains and lack of suitable media for screening was two reasons why the number of compounds identified frommyxobacteria is much less compared with thosefound in actinomycetes and fungi.Myxobacterial secondary metabolites exhibit many unique structural features andnovel modes action, making them attractive and promising sources for drugdevelopment such as Antibiotic, antimalarial, immunosuppressive, antiviral andinsecticidal activities

- Myxobacteria was the source of several new antibacterial: e.g., thuggacins, crocacin, indiacens, disciformycins, coralmycins, etc

- Several new antifungal compounds: e.g., pedein A and B, aurafurons Aand B, miuraenamides and cyrmeninshave been reported

- Several secondary metabolies anticancer agents are promising: Epothilone A and B, ixabepilone, Tubulysins, vincristine, nannocystin, Argyrin A andcruentaren A

Themany different compoundsfrom myxobacteriashow quite different mechanisms

of action Example: there are inhibitors ofprokaryotic (myxovalargin)and eukaryotic (gephyronic acid) proteinsynthesis, compounds that stimulate potassiumexport from Gram-positive bacteria (tartrolon) and compounds that bind to DNA (saframycin),interrupt DNAreplication and transcription (leupyrrinA), disruptthe integrity of the

cell membrane (ambruticin, jerangolid), inhibit acetyl CoA carboxylase activity (soraphen A) and and block the electron flow in the respiratory chain (stigmatellin A) [26], [20].

Some strains ofmyxobacteria isolated from soil have been published [20]

Table 1.2 A list of published bioactive compounds

Note: An: Angiococcus, Cb: Cystobacter, Cc: Corallococcus, Mx: Myxococcus,

Na: Nannocystis, Sg: Stigmatella, and So: Sorangium

1.4 ISOLATION AND IDENTIFICATION

1.4.1 Isolation of myxobacteria

The myxobacteria are found nearly everywhere; it very common and seems to be aunbiquitous group of organisms that can inhabit very diverse habitats Soil is thetypical habitat of myxobacteria

16 Nguyen Thi Kim Hong

Myxobacteria has two groups: (1) The cellulolytic myxobacteria; (2) The bacteriolytic myxobacteria Depending on the species, it is possibleto determine theappropriate isolation method The isolation of cellulolytic and bacteriolyticmyxobacteria was conducted using methods described by Reichenbach andDworkin (1992) [15].

1.4.1.1 Isolationofthe bacteriolytic myxobacteria

Escherichia coli (E coll) isa favorable substrate formyxobacteria Therefore, itwaschosen as a bait in baiting technique for isolation ofthe bacteriolytic myxobacteria Living E coli was spread into a cross-shaped on the wcx (the WAT medium add

25 mg/ml ofcycloheximide) Then, each soil sample was puton the tops and center

ofthe streaksE coll (five places persoil sample) [6]

1.4.1.2 Isolationof cellulose - decomposers strains

The ST21CXmedium (including the ST21 mediumadd 25 mg/ml of cycloheximide)with filter paper was chosen for isolation of the cellulolytic myxobacteria The cellulose is applied in the form of a sterile filterpaper, which isplaced on top oftheagar surface (four places) A pea-sized amount of soil sample will be placed on thefilter paper, incubatedat30°C After 10-20 days, thecellulolytic myxobacteria strains (specials genus Sorangium) showedunequivocal cellulose decomposition [6]

1.4.1.3 Isolationfromthe soil by natural substrates

The dung of various animals, especially herbivores such as rabbits, hares, deer,moose, sheep, and goats, are an excellent substrate for myxobacteria Rabbit dung was the preferred source of the early myxobacteria investigators and appeared to serve as natural bait, special fresh dung Strains of myxobacteria isolated were more abundant on soil containing fresh manurethan had not

Figure 1.8 Wild rabbit dung

About 5-6 autoclaved dung pellets from wild rabbits were placed on the soil, which was moistened with the mixture cycloheximide and amphotericin B Incubate thePetri dishes for 7-10 daysto allow the fruiting body to develop on the surface ofthepellets [6].

1.4.2 Purification of isolates

Myxobacteria have specific characteristics which do not disperse quickly in liquidmedia, but can move on solid mediumby gliding and slime production, easily carrycontaminants like fungi, soil amoebae, pretty hard to purification the isolates So, amedium containing an enzymatic hydrolysate ofcasein (CEH medium 1% w/v) hasbeen used for purification and purity tests combined in a single step

First, the isolates will be purified by two methods: (1) Transfer to fresh plates and/or(2) Using a mixture of antibiotics (ABI,AB3) And then, checkthepurifythe isolates

by transferring to CEH medium and shaking for 1-2 days at 30°C If the suspension was apparent after growth, consisting only of sediment, the culture was consideredpure [27],

1.4.3 Identify myxobacteria strains

1.4.3.1 Identify based onmorphological characteristics of myxobacteria

a Swarmcolonies

On media with relatively low nutrient contents (like VY2 medium), colonies ofmyxobacteria spread over the agar surface, called “swarms” The swarming behavior

of myxobacteria was different between strains

- Swarms (e.g., on VY/2 agar) tend to remain thin, film-like, and often show astriking surface structure in the form of radial veins and fields of tiny ridges

or waves Swarm edge usually has delicate fringes and flame-like protrusions(suborder Cystobacterineae)

- Swarms (e.g., on VY/2 agar) tend to sink into and to penetrate the agar, sometimes to the bottom of the plate; they usually form shallow pitsand bowl­like depressions; their surface structure is often less pronounced, although radial veins, ring-shaped ridges, and fanlike structures may be produced The agar surface in the swarm area may be more or less corroded (suborderSorangiineae)

18 Nguyen Thi Kim Hong

- Swarm colonies without a slime sheet, on certain media tend to corrode theagar plate very deeply, transform it into a spongy mass (suborderNannocystineae) [19].

b Fruiting body

The color of fruiting bodies may be white, brown or bright yellow, orange, red orlilac and size varies between 10 and 100pm This can be seen withthe naked eye and are easy to recognize under a dissecting microscope The fruiting body ofMyxobacteria has very diverse shapesand makesthe difference between strains [28],

- Myxococcus genera: spherical, solitary, visible,yellow, nacarat, brown;

- Cystobacter genera: without stipe, chained, or assembled, ovoid, brown;

- Corallococctts genera: in acluster, small, without stipe, nacarat;

- Nannocystis genera: sunk into medium, indistinct fruiting body structure;

- Polyangium genera: spherical, clump or solitary, orange, yellow, light brown;

- Sorangium genera: small, suck into medium, solitary, colourless

1.4.3.2 Identify based on the 16S rDNA sequencing

The genomic DNA was extracted and amplified in the PCR reaction The PCRproducts were purified with PCR products purification kit and sequenced at theprofessional organizations The 16S rRNAgene sequences of isolates were compared

to a cultured species database via BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and in order to identify different bacteria[28]

1.5.1 Antioxidant agents and myxobacteria

Free radicals are derived from normal metabolic processes in the human body orexternal sources such as ƯV exposure in sunlight, smoking, air pollutants, andindustrial chemicals Free radical formation occurs continuously in the cells as aconsequence of intracellular reactions The high concentrations of free radicals

promote oxidation, damage cellular structures, or biologically relevant molecules such as DNA, proteins, carbohydrates, and lipids Free radicals attack essential macromolecules leading to cell damage and degenerative diseases, cardiovascular disease, atherosclerosis, hypertension, diabetes, arthritis, weakened immune system, and the phenomenon of premature aging An antioxidant is a molecule that can neutralize free radicals through their free radical scavenging property, thus reducing its capacity for damage The study ofsecondary compounds possessing antioxidantcapabilities in myxobacteria facilitates the further approach of anticytotoxic effects

in this potential group of microorganisms [13]

1.5.2 Folin-Ciocalteu assay for polyphenols total

The total phenolic content was determined by the Folin-Ciocalteu (FC) colorimetricmethod A UV-vis spectrophotometer measured the absorbance at the wavelength of

765 nm, 760 nm, 750 nm, and 725 nm In an alkaline medium (like Na2COa/NaHCO3), phenolic compounds undergo autoxidation, and electrons aretransferred from them and other reducing agents to the molybdenum to form thecharacteristicblue complexes TheFC assay is often stated to measure "total phenolic content," but is not specific for phenolic compounds, and many non-phenoliccompounds can also reduce it, e.g., aromatic amines, sulfur dioxide, ascorbic acid, Cu(I), Fe(II) [10], [16],

1.5.3 ABTS radical cation scavenging activity

ABTS assayinvolves the direct productionof theblue/green ABTS‘+ (2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) through the reaction between ABTS andpotassium persulfate

Figure 1.9 The reaction mechanismof2,2’-azinobis(3-ethylbenzothiazoline-6-

sulfonic acid) with antioxidant

20 Nguyen Thi Kim Hong

Like DPPH assay, both methods apply decolorization assays to monitor the decrease

in their absorbance at characteristic a wavelength (DPPH and ABTS radicals respectively absorb at 517 and 734 nm) during the reaction This is an assay for theTrolox equivalent antioxidative capacity (TEAC), which was based onthe ability ofdifferent substances toscavenge theABTS radical cation in comparison to a standard (Trolox) In principle, the extent ofdecolorization equivalent percentage inhibition

ofthe ABTSradical cationis determined as a functionof concentrationand time andcalculated relative to Trolox's reactivity as a standard, under the same conditions

CHAPTER 2 MATERIALS AND RESEARCH METHODS

2.1 SUBJECTS AND MATERIALS

2.1.1 Subjects

Soil samples were collected from cities and provinces inVietnam: Ho Chi Minh City,Vinh Long, Hau Giang, Binh Duong, Thanh Hoa, Binh Phuoc, Binh Thuan, Ninh Binh, LongAn, Tien Giang, e.g

Soil samples were collected from the basement at a depth of 10 cm into clean, dry, and sterile falcons or bags The soil samples were air-dried as soon as possible toreduce the natural moisture ofsoil substrates and inhibit the development of fungi and worms

Eight strains of myxobacteria (VL3.1, VL 3.3, VL4.5, VL4.7, HG2.8, HG2.12,HG2.23, HG2.25) were obtained from the results of the thesis “Isolation ofMyxobacteria with biological activity from the soil” of Ms Nguyen Le Bao Ngoc in2019

2.L2 Instruments and equipments

Table 2.1 Listof laboratory instruments used in this research

3 Eppendorfcentrifuge Z206A Hemler, Germany

4 Cooling centrifuge Supra R12 Hand, Korea

6 Microbiological incubator INB 110 Memmert, Germany

9 Stereomicroscope - PRO view SZN6 Optika, Italia

10 Ultra-low temperaturefreezer MDF U33V Panasonic, Japan

13 Vertical laminar airflow cabinet - Aeolus V Telstar, UK

18 Universal Microplate Reader Biotek, USA

22 Nguyen Thi Kim Hong

19 Vacuum evaporator Heidolph, Germany

Equipments: Petri dishes, micropipettes (10-100 pl, 100-1000 pl, 1-10 ml); pipettetips, alcohol burners, filters, falcon, test tubes, aseptic syringes, sterile filters, duran(100 ml, 250 ml, 1000 ml); Erlenmeyer flasks (50 ml, 100 ml, 250 ml); Becher (50

ml, 100 ml, 250 ml, 500 ml), e.g

2.1.3 Media and chemicals

Media: WAT agar (WCX), ST21 agar, VY2 agar, EBS broth, CEH broth, p broth,VY3 broth, e.g

Chemicals: Most chemicals components were procured from Sigma-Aldrich(Germany), Himedia (India), Merck, e.g

Table 2.2 Some chemicals and reagents are used throughout the process

Resin: Amberlite XAD-16N adsorbent resin

Reagent: Folin-Ciocalteu reagent (FCR), 2,2-diphenyl-l- picryhydrazyl (DPPH), 2,2A-Azinobis(3-ethylbenzthiazoline)-6- _ sulfonic acid (ABTS);

2.2 RESEARCH METHODS

Figure 2.1 The study procedure

2.2.1 Samples collection and storage conditions

Soil samples were collected in areas less impacted by humans (no fertilizer use), nearlarge stumps such as orchards, straw, rice fields, e.g (near roots find strains with cellulose resolution) Soil samples were taken at a depth of 10 cm from the surface.The samples were dried in the air immediately after collection and stored at roomtemperature Otherwise, the sample can createfavorable conditions formold growth,negatively affectingfuture isolation

Soil samples were collected aseptically from 10 cm depth usingclean, dry, andsterilepolythene bags The samples were taken immediately to the laboratory, then weredried in 5 days under sunlight and crushed small After treatment, this soil used fordirect isolation of myxobacteria [4], [5], [6],

2.2.2 Treatment of soil samples

Dry soil samples were crushed to smaller About 25-30 g of soil was contained in falcon 50 ml (using wild rabbit dung method) or 5-10 g of soil in the method using

24 Nguyen Thi Kim Hong

the wcx medium/ ST21 medium Two methods willtreat soil samples: (1) Heating

in the water bath at 80°C, and/or (2) Using antifungal agents with cycloheximidesolution 100 pg/mL and amphotericin B 10 pg/mL

Freshly collected soil samples may be processed directly for isolation If samplescannot be processed immediately, they should be kept dry at room temperature forabout 2-4 weeks and could be capped in plastic containers or bags Because moistand wet samples promote the growth of variety of organisms, including theunwantedand difficult-to-treatfilamentous fungi

2.2.3 Isolation of myxobacterial strains

2.2.3.1 Isolation from natural substrates

We using the natural substrates that was wild rabbit dung Dry soil samples werecrushed to smaller About 25-30 gofsoil were contained in falcon 50 ml, and which were moistened by cycloheximide and amphotericin B for 15-20 minutes After that, the falcon will be steamed at 80°C for 20-30 minutes Let the falcon horizontal,submerged, flip over it every 10 minutes to heat evenly in the tube

The soil added to fill the Petri dish, about 4-5 autoclaved dung pellets from wild rabbits were placed, then cycloheximide solution 100 pg/mL and amphotericin B 10 pg/mL were added to reduce the development of fungi until sufficient moisture Check the bottom ofthe Petri dish to see if the soil is wet enough The Petri disheswere wrapped with paraffin and incubated at 30°C for 7-14daysto allowthe fruitingbody to develop on the surface ofthe pellets every day

Soil dishes were observed on a stereomicroscope daily to find out the appearance ofmyxospores If the lid of the Petri dish is difficult to observe by excessive moisture, dry the Petri lid on alcohol burners and close it oruse an alcohol lamp to dry the lid, then drywith an alcohol lamp or replace the other steamedPetri lid (Figure 2.3a) [6].2.2.3.2 Isolationofthe bacteriolytic myxobacteria

Escherichia coll (E coll)cells were culturedon LuriaBertanibroth (LB)medium for

24 hours Then, a sufficient amount of E coll suspensionwas suckedinto Eppendorf

2 ml and centrifuged for 1 minute at 13,000 rpm After that, the supernatant was removed, andthe E coll suspensionwas washed with sterile distilledwater to have E

coli biomass that was used as bait