chapter 6 antimicrobial and enzymatic activities of
TRANSCRIPT
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
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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).
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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
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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.
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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
IF2 Streptomyces fradiae A. florea Brood cells 2007
IF3 Streptomyces fradiae A. florea Brood cells 2007
IF4 Streptomyces fradiae A. florea Brood cells 2007
IF5 Streptomyces fradiae A. florea Brood cells 2007
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
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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
IT26 Streptomyces ambofaciens T. fuscobalteata Brood cells 2007
IT27 Streptomyces ambofaciens 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
IP5 Streptomyces rochei A. mellifera Pollen 2009
IP6 Streptomyces rochei 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
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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
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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
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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,
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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
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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
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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
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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
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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
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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 ± + ± + -
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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 +++ ++++ - + -
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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 - - + -
IT26 Streptomyces ambofaciens - - + -
IT27 Streptomyces ambofaciens - - + -
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 - - + +
IF2 Streptomyces fradiae - - + +
IF3 Streptomyces fradiae + - + +
IF4 Streptomyces fradiae - - + +
IF5 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 - - - -