CHAPTER 6 ANTIMICROBIAL AND ENZYMATIC ACTIVITIES OF

CHAPTER 6

ANTIMICROBIAL AND ENZYMATIC ACTIVITIES OF ACTINOMYCETES

ASSOCIATED WITH BEES AND DETECTION OF BIOSYNTHETIC GENES

SEQUENCES

6.1 Introduction

Actinomycetes are Gram-positive bacteria with a high G + C DNA content (>

55 mol %) (Stackbrandt et al., 1997) which produce two kinds of branching

mycelium, aerial and substrate mycelium. Actinomycetes are the most widely

distributed group of microorganisms in nature and occur in both soil and aquatic

environments (Goodfellow and Williams, 1983). The actinobacteria are known to be

significant antibiotic producers with some 70 – 80 % of the isolated antibiotics having

been isolated from Streptomyces species (Berdy, 2005). Recently, the rate of

discovery of new antibiotics from actinomycetes from common habitats has slowed

down therefore novel antibiotics must be found from actinomycetes in unexplored

habitats. For example, actinomycetes associated with insects have been explored and

reported one study found that fungus-growing ants have a mutualistic association with

filamentous bacteria belonging to the genus Pseudonocardia. These bacteria promote

the growth of the fungal in vitro and produce a highly potent antibiotic that selectively

inhibits the growth of the garden parasite Escovopsis. (Cafaro and Currie, 2005;

Currie et al., 1999). In addition, a Streptomyces strain was isolated from a fungus

89

garden of the leaf-cutting ants (Acromyrmex octospinosus). This bacterial strain

produced antifungal, candicidin macrolides, these were highly active against the

fungal pathogen Escovopsis (Header et al., 2009).

Colonies of Apis mellifera have been threatened by parasites and pathogens,

including mites, protozoa, viruses, fungi and bacteria. In particular, American

foulbrood (AFB) is an extremely contagious disease of honey bee (Apis mellifera) that

is caused by a Gram-positive spore forming bacteria Paenibacillus larvae. (Williams,

2000). This disease causes significant economic losses to the beekeeping industry

worldwide and poses problems for prevention and control because the bacterial spores

are highly resistant to heat and desiccation for up to 35 years (Reynaldi et al., 2008;

Williams, 2000). The main method of prevention and treatment of P. larvae infected

colonies is with the use of the antibiotic oxytetracycline (Williams, 2000). However,

tetracycline resistant strains have already been reported in the USA, Canada and

Argentina (Alippi, 2000; Evans, 2003; Miyagi et al., 2000; Reynaldi et al., 2008).

Other antibiotics, such as tylosin and lincomycin, have also been successfully used to

control AFB, but there are still concerns that resistant strains may emerge or

contaminated residues may be remained in hive products (Gonzalez and Marioli,

2010). For these reasons, the search for new strategies to control AFB and other bee

diseases is necessary. In this study, bacteria associated with the honey bee were tested

for antimicrobial activity against honey bee pathogens and most were found to belong

to the genus Bacillus (Alippi and Reynaldi, 2006; Evans and Armstrong, 2006; Sabate

et al., 2009; Yoshiyama and Kimura, 2009). In addition, actinomycetes isolated from

the tested beehives showed poor antimicrobial activity against AFB and EFB

pathogens (Promnuan et al., 2009).

90

Enzymes produced by microorganisms have been used in agriculture, medical

treatment and various other industries. In addition, many actinomycetes, especially

Streptomyces can produce a variety of extracellular enzymes including amylase,

protease, cellulase, lipase and chitinase (Abramic et al., 1999; Gupta et al., 1995;

Jaradat et al., 2008; Narayana and Vijayalakshmi, 2008; Mehta et al., 2005; Watanabe

et al., 1999; Yang and Wang, 1999). These types of enzymes generated by

microorganisms associated with insects have also been reported. Actinomycetes

isolated from termite (Termitidae) guts belonging to the genera Streptomyces and

Micromonosopora showed cellulolytic activity (Pasti and Belli, 1985) and

lignocellulose degrading ability (Pasti et al., 1990). The gut microbial community of

the wood-boring longhorned beetle, Saperda vestita, was also explored. Cellulolytic

microorganisms were found and identified as Sphingobium yanoikuyae, Fusarium

culmorum and Penicillium crustosum (Delalibera et al., 2005). In longicorn beetles,

bacterial communities and their exo-enzyme producing properties found that

Actinobacteria and Grammaproteobacteria were the dominant xylanase producers in

the gut of beetles (Park et al., 2007). Bacteria isolated from the gut of Bombyx mori

were able to degrade cellulose, xylan, pectin and starch (Anand et al., 2010).

However, the enzyme production form actinomycetes associated with bees have not

previously been studied and reported.

For screening antimicrobial production, the classical and alternative methods

such as detection for biosynthesis genes were used. In classical method,

actinomycetes were cultivated on fermentation media and test for antimicrobial

activity but in alternative method, actinomycetes isolates were screened for

biosynthetic genes which control bioactive compounds production. Modular

91

polyketide synthase type I (PKS-I), polyketide synthase type II (PKS-II) and

nonribosomal peptide synthase (NRPS) have extensively been described as

responsible for the synthesis of broad range of structurally diverse secondary

metabolites in actinomycetes. Many of bioactive molecules produced by

actinomycetes (and other organisms) are the product of either polyketide synthase

(PKS) or nonribosomal peptide synthethase (NRPS). These structurally diverse

metabolites include among others antibiotics, antifungal, antitumor agents,

anthelmintics, immunosuppressants, anticholesterolemics, antiparasitics and natural

insecticides. The specific primers targeted to PKS-I, PKS-II and NRPS actinomycete

sequences have been previously designed and validated, supporting a quick detection

of the biosynthetic enzymes (Ayuso-Sacido and Genillound, 2005; Katela et al.,

1999). There are several studies of screening the biosynthesis genes in actinomycetes.

The polyketide genes and nonribosomal peptide synthetase were detected in

actinomycetes isolated from Lichens (Gonzalez et al., 2005) and Challenger Deep

sediment from the Mariana Trench (Pathom-aree et al., 2006).

This study focused on actinomycetes samples isolated from bee hives to

determine their ability to inhibit bee and human pathogens with the purpose of

exploring possible applications of biological control. Furthermore, extracellular

enzyme production was also evaluated to understand any possible function as an

addition to food digestion in the honey bee. In addition, the detection of three

biosynthetic genes sequences (PKS-I, PKS-II and NRPS) using specific primers were

evaluated.

92

6.2 Materials and methods

6.2.1 Incubation and preparation of the crude extract

Fifty bee hive actinomycete isolates were isolated from honey bees and

stingless bees as shown in Table 6.1. Each actinomycetes isolate was grown in

oatmeal broth (Shirling and Gottlieb, 1966), V8 broth (V8 canned vegetable juice

(Campbell) 200 ml; CaCO3 3 g and 800 ml of distilled water, pH 7.2) and glucose-

yeast extract; GYE (1 % of glucose and yeast extract and 100 ml of distilled water,

pH 7.2) broth on a rotary shaker at 30 ⁰C for 21 days. Then supernatant was then

collected by centrifugation at 6,000 rpm for 10 min.

Table 6.1 The actinomycetes isolated from honey bees and stingless bees used in this

study. Isolate

No. Species Name Bee species Source Year

IF1 Streptomyces fradiae A. florea Brood cells 2006





IC1 Streptomyces drozdrwiczii A. cerana Brood cells 2006

IC2 Streptomyces badius A. cerana Brood cells 2007

IC5 Streptomyces badius A. cerana Gut 2008

IC6 Streptomyces albidoflavus A. cerana Gut 2008

IM1-3 Nocardiopsis alba A. mellifera Brood cells 2006

IM4-1 Nonomuraea roseoviolacea A. mellifera Brood cells 2006

IM7-2 Nonomuraea roseoviolacea A. mellifera Brood cells 2006

IM17-1 Actinomadura apis A. mellifera Brood cells 2006

IM21-1 Streptomyces misawanensis A. mellifera Brood cells 2006

IM21-2 Streptomyces olivaceus A. mellifera Brood cells 2006

IM22-1 Streptomyces aurantiogriseus A. mellifera Brood cells 2006

IM22-2 Streptomyces scabiei A. mellifera Brood cells 2006

IT1 Streptomyces pseudogriseolus T. laeviceps Brood cells 2006

IT2 Streptomyces rochei T. laeviceps Brood cells 2006

IT3 Streptomyces drozdowiczii T. laeviceps Brood cells 2006

IT4 Streptomyces mutabilis T. laeviceps Brood cells 2006

IT5 Streptomyces mutabilis T. laeviceps Brood cells 2006

93

Table 6.1 The actinomycetes isolated from honey bees and stingless bees used in this

study (cont).

Isolate

No. Species Name Bee species Source Year

IT6 Streptomyces minutiscleroticus T. laeviceps Brood cells 2006

IT7 Streptomyces albus T. laeviceps Brood cells 2006

IT8 Streptomyces tosaensis T. laeviceps Brood cells 2006

IT11 Streptomyces albus T. laeviceps Brood cells 2006

IT12 Streptomyces malaysiensis T. laeviceps Brood cells 2006

IT21 Streptomyces ambofaciens T. fuscobalteata Brood cells 2007

IT24 Streptomyces mutabilis T. fuscobalteata Brood cells 2007

IT25 Streptomyces coalescens T. fuscobalteata Brood cells 2007



IT28 Streptomyces violaceoruber T. fuscobalteata Brood cells 2007

IP1 Streptomyces diastaticus subsp.

siastaticus A. mellifera Pollen

2009

IP2 Streptomyces rochei A. mellifera Pollen 2009

IP3 Streptomyces griseus A. mellifera Pollen 2009

IP4 Streptomyces griseus A. mellifera Pollen 2009



FA5-1 Streptomyces fradiae A. florea adult 2009

FA5-2 Streptomyces drozdowiczii A. florea adult 2008

FA5-4 Streptomyces setonii A. florea adult 2009

FA5-5 Streptomyces fradiae A. florea adult 2009

FA5-6 Streptomyces diastaticus A. florea adult 2009

DG1 Streptomyces carnosus A. dorsata gut 2008

DG2 Streptomyces carnosus A. dorsata gut 2008

MA4-1 Streptomyces albidoflavas A. mellifera adult 2008

TA4-1 Streptomyces sp. T. collina adult 2007

TA4-8 Streptomyces sp. T. collina adult 2007

6.2.2 Microbial pathogens

Paenibacillus larvae LMG 9820T, Melissococcus plutonius LMG 20360 T and

Ascosphera apis HL52 the causative agent of American foulbrood (AFB), European

foulbrood (EFB) and Chalkbrood disease, respectively were use as test

microorganisms. The selected human pathogenic microorganisms used in this study

were Bacillus cereus TISTR 687T, Staphylococus aureus TISTR 517

T, Serratia

94

marcescens DMST 21632T, Salmonella typhymurium DMST 562

T and Candida

albicans ATCC 10231T.

6.2.3 Preparation of test microorganisms

The AFB pathogen, P. larvae LMG9820T was grown in THClYGP broth

(Steinkraus and Morse, 1996) at 30 ⁰C for 5 days, M. plutonius LMG 20360 T was

grown in SYPG broth (Bailey and Ball, 1991) at 30 ⁰C under anaerobic conditions for

5 days and the bacterial human pathogens (B. cereus TISTR 687T, S. aureus TISTR

517T, S. marcescens DMST 21632

T and S. typhymurium DMST 562

T) were grown in

nutrient broth at 37 ⁰C for 1 day. C. albicans ATCC 10231T was grown in YM broth

at 37 ⁰C for 1 day and the fungus causing the Chalkbrood disease, A. apis HL52 was

grown on Sabouraud dextrose agar (SBA) at 28 ⁰C for 5 days.

6.2.4 Assay of antimicrobial activity

Antimicrobial activities of each actinomycete isolate against P. larvae

LMG9820T were tested using the agar well diffusion technique. Bacterial suspensions

of P. larvae LMG 9820T were adjusted to a 0.5 Mac Farland Standard. An aliquot of

0.1 ml of this solution was poured and spread on a Petri dish containing THClYGP

agar. Wells with a 6 mm diameter were made on THClYGP plates using a cork borer.

Thirty µl of cell free extract for each actinomycete isolate was then applied to one

well of each plate and 0.5 % w/v of gentamycin sulfate was used as positive control.

Oatmeal, V8 and GYE broth were used as negative controls. The plates were

incubated at 30 ⁰C for 3 days. For M. plutonius ATCC 35311T, C. albicans ATCC

10231T and some of the selected human pathogens, the same testing protocol were

95

used as previously described but SYPG agar, YM agar and nutrient agar were used

instead of THClYGP agar, respectively. M. plutonius ATCC 35311T was incubated at

30 ⁰C under anaerobic condition for 5 days. C. albicans ATCC 10231T and the human

pathogens were incubated at 37 ⁰C for 1 day. In the case of the Chalkbrood pathogen,

A. apis HL52 was grown on the center of a SBA plate for 5 days. Six mm diameters

wells were then made around the fungus colony. Nystatin (0.5 % w/v) was used as

positive control and the plates were incubated at 28 ⁰C for 3 days. The diameters of

the inhibition zones were measured and the mean and SD were calculated form

triplicate experiments.

6.2.5 Screening for extracellular enzyme production

Fifty bee hive actinomycete isolates were evaluated for their ability to produce

extracellular enzymes using the plate screening method. Each actinomycetes was

grown on a starch agar, skim-milk agar, carboxymethylcellulose (CMC) agar (Kasana

et al., 2008), Tributyrin agar (Anderson, 1939) and colloidal chitin basal (CCB) agar

to screen for amylase, protease, cellulose, lipase and chitinase, respectively. The

plates were incubated at 30 ⁰C for 5 days. The protease, chitinase and lipase activity

were observed as a clear zone diameter around the colonies and measured. For the

detection of amylase activity, the plates were flooded with Lugol solution for 1

minute and then the iodine was poured off of the plates. For the observed cellulose

production, the plates were flooded with 0.1 % Congo red for 15-20 minutes and then

with 1M NaCl for 15-20 minutes (Teather and Wood, 1982). The diameters of the

96

clear zones were then measured and the means and standard deviations were

calculated from triplicate experiments.

6.2.6 Screening for biosynthetic genes

Each actinomyceteisolate was grown in oatmeal broth at 30 ⁰C for 3 weeks.

Biomass of each actinomycete isolate was collected by centrifugation at 6,000 rpm for

10 minutes, and then washed with sterile distilled water (twice). All samples were

kept in -20 ⁰C for further study.

Genomic DNA of each isolate was extracted according to a modification of

Nakajima et al., (1999) and Sharma and Singh, (2004). The DNAs were stored at -20

⁰C for further studies.

Fifty actinomycete isolates were screened for the presence of biosynthetic

genes (type-I polyketide synthase (PKS-I), type-II polyketide synthase (PKS-II) and

nonribosomal peptide synthetase (NRPS) genes). K1F (5’-

TSAAGTCSAACATCGGBCA-3’) and M6R (5’-TACTGGTACSGSAACCTGCG-

3’) for detecting PKS-I sequences (Ayuso-Sacido and Genilloud, 2004); KSα (5’-

TSGCSTGCTTCGAYGCSATC-3’) and KSβ (5’-TCGCCBAAGCCGCCNAAGGT-

3’) for detecting PKS-II sequences (Katela et al., 1999) and A3F (5’-

GCSTACSYSATSTACACSTCSGG-3’) and A7R (5’-

STACCGSACSGGBGACSTS-3’) for detecting NRPS sequences (Ayuso and

Genilloud, 2004).

The PCR condition for the amplification of PKS-I, PKS-II and NRPS genes

were modified from Ayuso-Sacido and Gentlloud, 2005 and Katela et al., 1999. For

extracted DNA, about 10-20 ng of template, 2 µM of each primer, 200 µM of dNTP,

97

3 % dimethylsulfoxide (DMSO), 1.5 mM MgCl2, 2 units of Taq DNA polymerase

(Invitrogen) were used in 25 µl reaction volume. Amplification reactions were carried

out in MyCyclerthermal cycler (Bio-Rad).

The PCR temperature profile for PKS-I and NRPS was 5 min at 95 ⁰C, 30

cycles of 0.5 min at 95 ⁰C, 2 min at 55 ⁰C for K1F/M6R, 59 ⁰C for A3F/A7R, 4 min

at 72 ⁰C and a final extension step 10 min at 72 ⁰C. The PCR temperature profile of

PKS-II was: 5 min at 96 ⁰C, 30 cycles of 0.5 min at 95 ⁰C, 1 min at 60 ⁰C, 1.5 min at

72 ⁰C and a final extension step 10 min at 73 ⁰C. Amplification products were

analyzed by electrophoresis in 1 % (w/v) agarose gels stained with ethidium bromide.

6.3 Results and discussion

6.3.1 Antimicrobial activity against bee and some human pathogens

The inhibition zone of each supernatant from the three different culture media

was tested against the growth of each tested organism. Among the 50 actinomycete,

12 isolates were able to inhibit the growth of P. larvae LMG9820T (Fig. 6.1). The

actinomycete isolates IT12, IP5, DG1 and DG2 produced antimicrobial compounds in

oatmeal (ISP3) broth. The isolate IC6, IT5, IM21-1, IM21-2 and DG1 produced

antibiotics in V8 broth. GYE broth was suitable for antibiotic production of the strains

IP3, IM4-1, IM7-2, IM17-1 and DG1. Out of the 12 isolates, four exhibited strong

inhibitory activity against P. larvae LMG 9820T with inhibition zone diameters of

26.67 ± 1.53 - 28.00 ± 2.00 mm. when compared with 0.5 % w/v gentamycin (26.00 ±

1.73 - 26.33 ± 1.15 mm). The four actinomycete strains were identified as

Streptomyces malaysiensis (IT12), Streptomyces carnosus (DG1 and DG2),

Streptomyces olivaceus (IM21-2) and Actinomadura apis (IM17-1). The European

98

foulbrood and Chalkbrood pathogens were also tested with each supernatant obtained

from the bee hive actinomycetes. The results showed that none actinomycete isolates

were able to inhibit the growth of M. plutonius LMG 20360 T and A. apis HL52. In

addition, the human pathogens; Bacillus cereus TISTR 687T, Staphylococus aureus

TISTR 517T, Serratia marcescens DMST 21632

T, Salmonella typhymurium DMST

562T, Candida albicans ATCC 10231

T were also used as test organisms. Eight

isolates (IT12, IF3, DG1, DG2, IP2, IM4-1 IM7-2, and IM21-2) were found to have

an inhibitory effect on B. cereus TISTR 687T (8.00 ± 0.00 – 15.00 ± 1.00 mm). Two

isolates (IT12 and IP5) showed the ability to inhibit the growth of S .aureus TISTR

517T (8.00 ± 0.00 – 16.33 ± 0.58 mm). Only isolate IT12 was able to inhibit the

growth of C. albicans ATCC 10231T (11.00 ± 1.00 mm) and only the isolate IP2

showed an inhibitory effect against S. typhymurium DMST 562T (8.00 ± 0.00 mm).

However, none of the actinomycete isolates were able to inhibit the growth of S.

marcescens DMST 21632T.

According to several studies of microorganisms associated with the honey bee

(Apis mellifera) (Gilliam, 1997; Gilliam and Morton, 1978; Piccini et al., 2004; Rada

et al., 1997), numerous bacteria which have been isolated from the honey bee have

been investigated for potential use as antimicrobial agents against honey bee

pathogens. In particular, members of genus Bacillus, Brevibacillus,

Stenotrophomonas and Acinetobacter displayed antagonistic activity against P. larvae

(Alippi and Reynaldi, 2006; Evans and Armstrong, 2006; Yoshiyama and Kimura,

2009; Sabate et al., 2009). It is commonly known that actinomycetes are prolific

produces of a wind range of antimicrobial agents (Watve et al., 2001). However,

actinomycetes have not been widely studied for use to control bee pathogens. One

99

previous study found actinomycetes isolates which showed a small inhibition zone

against P. larvae LMG 9820T using the disc diffusion assay (Promnuan et al., 2009).

However, in this study we optimized the cultivating media (V8, oatmeal (ISP3) and

GYE broth) and demonstrated that each actinomycete isolate could produce

antimicrobial compounds against the AFB pathogen using a specific medium (Fig.1).

Only one isolate (DG1) produced antimicrobial compounds in all media. Out of 50

isolates, 12 isolates displayed significant inhibitory effects against the AFB pathogen

which were much higher than any previously reported. Amongst these isolates, the

four isolates which showed the highest inhibitory activity were identified as S.

malaysiensis, S. carnosus, S. olivaceus and A. apis, the latter of which was the recent

novel species described in the genus Actinomadura (Promnuan et al., 2011) and was

found to show an inhibitory effect as high as purified antibiotic, gentamycin. As a

result of this study, actinomycetes isolated from bee hives could be a potent candidate

in controlling the American foulbrood disease.

Fig. 6.1 Antimicrobial activity of bee hive actinomycetes tested on different culture

media (oatmeal, V8 and GYE broth) against the growth of Paenibacillus larvae LMG

9820T.

0

5

10

15

20

25

30

35

IT5

IT12

IP3

IP5

DG

1

DG

2

IC6

IM4

-1

IM7

-2

IM1

7-1

IM2

1-1

IM2

1-2

Gen

tam

yci

n

Inh

ibit

ion

zo

ne

(mm

)

Isolate Number

Oatmeal

V8

GYE

100

6.3.2 Extracellular enzyme production from actinomycetes

All of the actinomycete isolates were screened for extracellular enzyme

production. These results are shown in Table 6.2. Forty seven of the 50 isolates

produced amylase with three isolates; S. albidoflavus (IC6 and MA4-1) and S. griseus

(IP4) showing the greatest clear zone diameters (29.3 ± 2.1 - 33.7 ± 1.5 mm) on starch

agar. Forty three isolates displayed clear zones on skim milk agar with six isolates; S.

albidoflavus (IC6 and MA4-1), S. drozdowiczii (IT3 and FA5-2) and S. griseus (IP3

and IP4) showing proteolytic activity with clearing zone diameters of 32.0 ± 2.0 -

37.7 ± 0.6 mm. Forty isolates produced cellulase on the CMC agar. S. badius (IC5), S.

rochei (IT2, IP2, IP5 and IP6), S. drozdowiczii (IT3), S. violaceoruber (IT28), S.

griseus (IP5) and S. fradiae (IF3) presented with clear zone diameters in the range of

24.3 ± 4.0 - 28.0 ± 0.0 mm. Forty one isolates showed lipolytic enzyme activity on

Tributyrin agar, and S. rochei (IP5) presented the widest clear zone diameter of 26.3 ±

0.6 mm. The majority of the bee hive actinimycetes were not able to produce chitinase

and only fifteen isolates were found to poorly produce chitinase. Fifteen isolates of

the bee hive actinomycetes were found to produce extracellular enzymes; amylase,

protease, cellulase, lipase and chitinase which are shown in Fig. 2. Comparing among

these 15 isolates S. albidoflavus (IC6 and MA4-1), S. drozdowiczii (IT3, FA5-2) and

S. griseus (IP3 and IP4) were the most potent producers of proteases and amylases.

The actinomycetes were also evaluated for their ability to produce

extracellular enzymes including amylase, protease, cellulose, lipase and chitinase.

Most isolates were able to produce amylases and proteases which are likely to be

utilized in organic compounds which occur in bees/hives. From previous studies,

microorganism associated with the bee produced enzymes involve in protein, lipid

101

and carbohydrate metabolism (Gilliam et al., 1989). However, in our study, cellulase,

lipase and especially chitinase were only poorly produced. When compared with

actinomycetes associated with termites (Termitidae) gut. The actinomycetes

belonging to the genera Streptomyces and Micromonosopora showed cellulolytic

(Pasti and Belli, 1985) and lignocellulose degrading abilities (Pasti et al., 1990)

indicating that these bacteria might help with cellulose and lignocelluloses digestion

in the Termites gut. In the honey bee and stingless bee, the food sources are mostly

pollens and nectar therefore, the some microbial groups that dominate in the bees/bee

hives may not be required to help in food digestion.

Further studies are needed to characterize the chemical nature of the

compounds implicated in the inhibitory activities and also clarify the functional role

of these actinomycetes in bee.

Fig. 6.2 Comparison of fifteen actinomycetes isolates with a strong ability to produce

the extracellular enzymes; amylase, protease, cellulase, lipase and chitinase.

0

5

10

15

20

25

30

35

40

45

Cle

ar

zon

e d

iam

eter

s (m

m)

Isolate Number

Amylase Protease Cellulase Lipase Chitinase

102

102

Table 6.2 Screening for enzyme production from actinomycetes isolated from bees. Clear zone diameters (mm) ≤ 6.0; -, 6.1-12.0; ±,

12.1-18.0; +, 18.1-24.0; ++, 24.1-30.0; +++, 30.1-36.0; ++++ and 36.1-42.0; +++++.

Strain no. Species Name Amylase Protease Cellulase Lipase Chitinase

IC1 Streptomyces drozdrwiczii + ++ ++ - -

IC2 Streptomyces badius ++ +++ ++ ± -

IC5 Streptomyces badius +++ +++ +++ ± -

IC6 Streptomyces albidoflavus ++++ +++++ - + -

IT1 Streptomyces pseudogriseolus +++ + + ± ±

IT2 Streptomyces rochei ± ++ +++ ++ ±

IT3 Streptomyces drozdowiczii +++ ++++ +++ ± -

IT4 Streptomyces mutabilis + ++ + ± -

IT5 Streptomyces mutabilis ++ ++ + ± -

IT6 Streptomyces minutiscleroticus ++ + ± ± -

IT7 Streptomyces albus + + + ± -

IT8 Streptomyces tosaensis ± - - ++ -

IT11 Streptomyces albus + + + ± -

IT12 Streptomyces malaysiensis ± ++ ++ ± -

IT21 Streptomyces ambofaciens + ++ ++ + ±

IT24 Streptomyces mutabilis + + - - -

IT25 Streptomyces coalescens ± - ++ ++ ±

IT26 Streptomyces ambofaciens + ++ ++ + -

IT27 Streptomyces ambofaciens + +++ ++ + ±

IT28 Streptomyces violaceoruber ± ++ +++ + ±

IP1 Streptomyces diastaticus subsp. siastaticus + ++ + ± -

IP2 Streptomyces rochei ± ++ +++ ++ ±

IP3 Streptomyces griseus ++ ++++ - - -

IP4 Streptomyces griseus ++++ +++++ - + -

IP5 Streptomyces rochei ± +++ +++ +++ ±

IP6 Streptomyces rochei ± ++ +++ ++ ±

FA5-1 Streptomyces fradiae ± + ± + -

103

103

Table 6.2 Screening for enzyme production from actinomycetes isolated from bees. Clear zone diameters (mm) ≤ 6.0; -, 6.1-12.0;

±, 12.1-18.0; +, 18.1-24.0; ++, 24.1-30.0; +++, 30.1-36.0; ++++ and 36.1-42.0; +++++ (cont.).

Strain no. Species Name Amylase Protease Cellulase Lipase Chitinase

FA5-2 Streptomyces drozdowiczii +++ ++++ ++ ± -

FA5-4 Streptomyces setonii ++ - ++ ± -

FA5-5 Streptomyces fradiae ± +++ + + -

FA5-6 Streptomyces diastaticus + +++ + ± ±

IF1 Streptomyces fradiae ± + ++ + -

IF2 Streptomyces fradiae ± ++ ++ + -

IF3 Streptomyces fradiae ± +++ +++ + -

IF4 Streptomyces fradiae ± + ± + -

IF5 Streptomyces fradiae ± + ++ + ±

TA4-1 Streptomyces sp. ± ± ± - -

TA4-8 Streptomyces sp. ± ± ± - -

IM1-2 Streptomyces lanatus ± ± - - -

IM1-3 Streptomyces lanatus ± ± - - -

IM4-1 Nonomuraea roseoviolacea - - ± ± -

IM7-2 Nonomuraea roseoviolacea - - + + ±

IM17-1 Actinomadura apis - + - - -

IM21-1 Streptomyces misawanensis ++ +++ ± ± -

IM21-2 Streptomyces olivaceus ++ +++ ++ ± ±

IM22-1 Streptomyces aurantiogriseus ± ± - - -

IM22-2 Streptomyces scabiei ++ - ++ ± -

DG1 Streptomyces carnosus +++ +++ ± ± ±

DG2 Streptomyces carnosus +++ - ++ ± ±

MA4-1 Streptomyces albidoflavas +++ ++++ - + -

104

104

6.3.3 Detection of biosynthetic genes

The fifty actinomycete isolates were screened for the presence of biosynthetic

genes; PKS-I, PKS-II and NRPS genes using three pairs of specific primers. Out of 50

isolates, PKS-I, PKS-II and NRPS sequences, it was found that 2 isolates (4 %), 30

isolates (60 %) and 19 isolates (38 %) were positives for the tested genes respectively.

Most isolates were belonging to genus Streptomyces, the results are shown in Table

6.3. The biosynthetic genes were found in some Streptomyces spp.

Nowadays molecular screening strategies were used to discover new bioactive

compounds from actinomycetes, especially polyketides and peptide antibiotics using

primers synthesized from PKS and NRPS genes (Ayuso and Genilloud, 2004; Bull et

al., 2000; Metsa-Katela, 2003; Savic and Vasijevic, 2006). However, the presence of

biosynthetic systems did not relate to the production of antimicrobial activities. Some

actinomycete isolates both of Streptomyces and other genera, which showed

antimicrobial activities in plate screening method but biosynthetic genes could not be

detected. This result may explain that (i) the PCR amplification condition and primers

were not specific for these actinomycete species/strains; (ii) they might produce other

types of antimicrobial compounds such as ribosomally synthetised peptides with the

biological activity or bacteriocins (Arias et al., 2011).

On the other hand, some actinomycete isolates which did not produce

antimicrobial compounds on screening plates, contained biosynthetic genes. This

result confirmed that many potential poyketides and peptide compounds are missed

during prior screening. It is possible that in the limited of cultivation conditions, the

biosynthetic genes in some actinomycetes isolates were not well expressed, so the

richness of their genomes in respect of the biosynthetic gene does not always reflect

105

105

in the production of antimicrobial metabolite observed by classical screening. In

addition, the genome analysis of actinomycetes has revealed the presence of

numerous silent or cryptic gene clusters encoding putative natural products. These

loci remain dormant until appropriate chemical and physical signals that could induce

their expressions. These suggest that these actinomycete strains may produce a greater

number of bioactive compounds than has been detected by fermentation broth

analysis (Zazopoulos et al. 2003).

According to the study from Ayuso and Genilloud (2004), 210 actinomycetes

of 32 genera were detected for NRPS and PKS-I sequences using degenerate PCR

primers. Both NRPS and PKS-I sequences were detected from Streptomyces fradiae

ATCC 6855T, S. fradiae DSM 4175

T, S. albidoflavus ATCC 25422

T, S. griseus

ATCC6855T, S. ambofaciens ATCC 23877

T, S. violaceoruber ATCC 14980

T and S.

diastaticus ATCC 3315T. In this study, only NRPS sequence was detected from S.

fradiae, S. albidoflavus and S. griseus. In addition, neither PKS-I nor NRPS sequence

was detected from S. ambofaciens, S. violaceoruber and S. diastaticus. The results

indicated that our actinomycete strains have some genetic variation from the type

strains, and therefore, the degenerated primers for PKS-I were not specific for the

target site. For better understanding, cloning for the NRPS sequences prior to

sequencing is necessary to investigate this further.

106

106

Table 6.3 Detection of biosynthetic genes in actinomycetes isolated from bees.

Str

ain

no

.

Species Name

Pla

te

scre

enin

g

met

ho

d*

PK

S-I

am

pli

fica

tio

n

PK

S-I

I

am

pli

fica

tio

n

NR

PS

am

pli

fica

tio

n

IC1 Streptomyces drozdrwiczii - + + +

IC2 Streptomyces badius - - - -

IC5 Streptomyces badius - - - -

IC6 Streptomyces albidoflavus + - + +

IT1 Streptomyces pseudogriseolus - - + -

IT2 Streptomyces rochei - - - +

IT3 Streptomyces drozdowiczii - - - -

IT4 Streptomyces mutabilis - - + -

IT5 Streptomyces mutabilis + - + -

IT6 Streptomyces minutiscleroticus - - + -

IT7 Streptomyces albus - - + -

IT8 Streptomyces tosaensis - - + +

IT11 Streptomyces albus - - + -

IT12 Streptomyces malaysiensis + - + -

IT21 Streptomyces ambofaciens - - + -

IT24 Streptomyces mutabilis - - + -

IT25 Streptomyces coalescens - - + -



IT28 Streptomyces violaceoruber - - - -

IP1

Streptomyces diastaticus subsp.

diastaticus

- -

-

-

IP2 Streptomyces rochei + - + +

IP3 Streptomyces griseus + - + +

IP4 Streptomyces griseus - - + +

IP5 Streptomyces rochei + - + +

IP6 Streptomyces rochei - - + +

FA5-1 Streptomyces fradiae - - + +

FA5-2 Streptomyces drozdowiczii - + + +

FA5-4 Streptomyces setonii - - - +

FA5-5 Streptomyces fradiae - - + +

FA5-6 Streptomyces diastaticus - - - -

IF1 Streptomyces fradiae - - + +


IF3 Streptomyces fradiae + - + +



TA4-1 Streptomyces sp. - - - -

TA4-8 Streptomyces sp. - - - -

107

107

Table 6.3 Detection of biosynthetic genes in actinomycetes isolated from bees

(cont.). S

tra

in n

o.

Species Name

Pla

te

scre

enin

g

met

ho

d*

PK

S-I

am

pli

fica

tio

n

PK

S-I

I

am

pli

fica

tio

n

NR

PS

am

pli

fica

tio

n

IM1-2 Streptomyces lanatus - - - -

IM1-3 Nocardiopsis alba - - - -

IM4-1 Nonomuraea roseoviolacea + - - -

IM7-2 Nonomuraea roseoviolacea + - - -

IM17-1 Actinomadura apis + - - -

IM21-1 Streptomyces misawanensis + - - -

IM21-2 Streptomyces olivaceus + - - -

IM22-1 Streptomyces aurantiogriseus - - - -

IM22-2 Streptomyces scabiei - - - +

DG1 Streptomyces carnosus + - + -

DG2 Streptomyces carnosus + - + -

MA4-1 Streptomyces albidoflavas - - - -

Documents

CHAPTER 6 ANTIMICROBIAL AND ENZYMATIC ACTIVITIES OF