ISSN 08914168, Molecular Genetics, Microbiology and Virology, 2013, Vol. 28, No. 1, pp. 3239.

Allerton Press, Inc., 2013.

EXPERIMENTAL WORKS

Screening for Type I Polyketide Synthases Genes of Endophytic

Streptomycetes Isolated from Parthenium hysterophorus L.1
R. Tanvir, I. Sajid, and S. Hasnain
Department of Microbiology and Molecular Genetics, University of the Punjab,
QuaideAzam Campus, 54590, Lahore, Punjab Pakistan
email: shahida@mmg.pu.edu.pk
Received July 1, 2012

AbstractPolyketides are a vital group of secondary metabolites comprising of antifungal, antibacterial as

well as anticancer agents. The multifunctional enzymes responsible for the biosynthesis of these secondary
metabolites are polyketide synthases (PKS) in which polyketide synthases type I (PKSI) is mainly responsi
ble for highly assorted group of metabolites with substantial medical importance. In this study molecular
screening was done using six sets of degenerate primers to determine the presence of PKSI gene cluster in
endophytic Streptomyces isolated from the weed Parthenium hysterophorus L. A total of 40 endophytes were
isolated and identified by morphological, biochemical and physiological characterization as belonging to the
genus Streptomyces. The 16S rRNAgene sequencing of the selected isolates exhibited maximum similarity
with different species of Streptomyces such as Streptomyces rochei (99%), Streptomyces litmocidini (99%),
Streptomyces enissocaesili (99%), Streptomyces djakartensis (99%), Streptomyces olivaceus (99%), Streptomy
ces spp. (99%), Streptomyces plicatus (99%), Streptomyces geysiriensis (99%) and Streptomyces vinaceusdrap
pus (99%). The molecular screening revealed the presence of PKSI gene with a PCR amplification products
of size ~300 bps, ~320 bps and ~700 bps in the isolates RT13, RT43, RT47, RT49, RT54, RT56, RT57,
RT58, RT59, RT61 and RT65 through all the six sets of primers used. To the best of our understanding
no previous study has been carried out reporting the molecular screening of PKSI genes in endophytic Strep
tomyces from Parthenium hysterophorus L. The results provide an insight into an unexplored environment
containing endophytic Streptomyces harboring the polyketide synthases gene which if further investigated
may lead to a new source of antimicrobial agents.
Keywords: endophytes, Parthenium hysterophorus L., Streptomyces, polyketide synthases type I (PKSI)
DOI: 10.3103/S0891416813010060

INTRODUCTION

a complex pathway involving many other genes for

modification of products and synthesis of precursors
[13]. On the basis of the molecular architecture the
PKSs are classified into 3 main types [13]. Type I
polyketide synthases (PKSI) containing multiple
domains are multifunctional enzymes and are quite
large, structured into modules where each module is
responsible for a cycle of chain elongation [6]. This
polyketide synthases type I and also the nonriboso
mal peptide synthetases (NRPSs) are involved in the
biosynthesis of natural products that are in preclinical
pharmaceutical progress for e.g. epothilone and
cryptophycin, or that are currently in use like rapamy
cin, bleomycin, and FK506 [2].

Polyketides produced by actinobacteria belong to a

vital group of secondary metabolites [16] including
macrolides, polyethers and aromatics which exhibit
various biological activities such as antifungal, anti
bacterial, immunosuppressive, antitumor and antipar
asitic effects. They are a prosperous sources of clini
cally valuable pharmaceuticals, [6] cholesterol lower
ing drugs, antitumor, and anticancer agents [3].
During the past decade, the number of sequenced and
cloned genes in the polyketide biosynthesis has aug
mented rapidly enabling the possibility of the discov
ery of novel compounds [8]. Polyketides are a conse
quence of the successive condensation of carboxylic
acid units likewise to fatty acids yet they can be built up
by numerous permutations of the steps of fatty acid
biosynthesis from more than one type of monomer.
Polyketide synthases (PKSs) are the main enzymes
requisite for polyketide synthesis functioning analo
gously to fatty acid synthases that only forms a part of
1

The PKS type II is made up of domains as individ

ual proteins that mutually manage in trans polyketide
formation, each domain in this case is used a number
of times for a single polyketide chain elongation. The
PKS type III or the chalcone synthases, are ACP
independent made up of a single KS protein catalyzing
numerous decarboxylase condensations [15].

The article is published in the original.

32

SCREENING FOR TYPE I POLYKETIDE SYNTHASES GENES

Due to the current molecular approaches the pres

ence of a large number of cryptic PKS genes has been
successfully exposed. For example, Streptomyces spp.
genome analysis has verified that their strains hold
many PKS genes with unidentified functions. Thus,
Streptomyces appear to have the potential to produce
thus far undisclosed polyketides. The sequencing of
PKS genes is frequently used as a screening method for
the detection of novel polyketides since their occur
rence in organisms provides good clues for manufac
ture of novel polyketides [6].
The aim of the present study was the molecular
screening for PKS type I gene sequences in endo
phytic Streptomyces isolated from P. hysterophorus
using the degenerate primers selected for this purpose.
Since PCR screening has been done on Streptomyces
isolated from numerous plants but to the best of our
knowledge no screening has been done on Streptomy
ces isolated from a weed plant therefore this work may
result in screening of undisclosed polyketides of signif
icant biosynthetic potential. However, the PKSI gene
sequencing has not been done in the study which once
carried out may further lead to identify putative PKS
genes from gene libraries of the endophytic Streptomyces.
MATERIALS AND METHODS
Sample collection. Healthy plants of Parthenium
hysterophorus were collected during the period of June
2009 to November 2009 from the agricultural lands at
the university campus. The plants were carefully
rooted and shifted to the lab in labeled sterile bags and
were processed within 4 hours after collection.
Isolation and characterization of endophytic strep
tomycetes. Plant samples were thoroughly washed
under running tap water in order to remove all soil par
ticles and cut into 0.5 cm segments. Surface steriliza
tion was done by the five step sequential process.
Briefly, the segmented tissues were immersed in 70%
ethanol for five minutes and then in 0.9% sodium
hypochlorite solution for twenty minutes. To com
pletely remove the disinfectants each of the tissue was
washed three times with autoclaved distilled water fol
lowed by dipping the tissue in 10%NaHCO3 for ten
minutes for disruption and inhibition of endophytic
fungal growth. Finally the tissue segments were
washed with autoclaved distilled water.
The tissue segments prepared were placed on acti
nomycetes isolation agar (Difco laboratories), Glyc
erol casein KNO3 agar [7] and Rice agar (rice boiled
for 30 min, then filtered to remove insoluble materials,
18.0 g/L agar was added, pH 7.6) and incubated at
28C for a period of 3 weeks. As a negative control,
plant tissue segments were aseptically rolled and
plated on the three media, and incubated at 28C. The
selected colonies were purified by repeated sub cultur
ing on glucose yeast extract malt extract agar (GYM)
[12] until pure cultures were obtained.

33

The isolates were identified on the basis of mor

phological (Table 1), biochemical and physiological
characteristics and 16S rRNA gene sequencing. Bio
chemical and physiological characterization which
included melanin production, utilization of nine dif
ferent sugars as carbon source was performed accord
ing to the conventional methods as described by
Shirling and Gottlieb, 1966 [12] (Table 2). Formation
as well as utilization of organic acids and oxalate,
hydrolysis of esculin, arbutin, urea and allantoin was
also determined (Table 3). 16S rRNA gene sequencing
was done commercially at the Macrogen sequencing
facility (Macrogen Inc., Seoul, Korea). The gene
sequence data obtained was first analyzed using the
advanced BLAST search program at the NCBI web
site: http://www.ncbi.nlm.nih.gov/BLAST/. The nucle
otide sequence data was deposited to GenBank, and
the Genbank accession numbers for 12 strains were
obtained. The number of nucleotides sequenced in
each case and the gene bank accession numbers for
selected strains are summarized in Table 4.
Isolation of genomic DNA. Genomic DNA of the
Streptomyces strains was isolated using the method
described by Sajid et al. 2009 [10]. Briefly, the strains
were inoculated in GYM broth and incubated at 28C
for a period of 1 week. Then aperally in the soil but
lately some species have been described in the rhizo
sphere of plant roots and in other plant tissue as well
[1], Despite the fact that the majority of such bacteria
are phyto pathogenic, a considerable number have
also been found that inhabit the plant without causing
any disease. Such colonizing bacteria are called endo
phytes [4]. A number of endophytic Streptomyces
strains were isolated from different tissues of P. hys
terophorus which is a common weed found in the ter
restrial areas of Pakistan. These endophytes were mor
phologically characterized and all the selected isolates
exhibited similar morphological features mainly the
appearance of a rough and dry colony texture with
variable colored substrate and aerial mycelium as
described in Table 1. The physiological and biochem
ical characterization was performed following the
methods adopted in International Streptomyces
Project (ISP)Whirling and Gottlieb [12] and the results
of these tests demonstrated soluble pigment production
for all the isolates except RT65. The sugar utilization
tests were also positive for majority of the isolates as
described in Table 2. Similarly, most the isolates also
exhibited positive results for utilization of organic
acids and hydrolysis of esculin and arbutin as depicted
in Table 3.
Natural product screening has always been focused
on the discovery of new bioactive metabolites and now
it is possible to directly detect the genes involved in the
synthesis of new secondary metabolites [14] which has
enabled the manipulation of polyketide biosynthetic
pathways to yield novel compounds [5]. Since to the
best of our comprehension the polyketide biosynthetic
potential of endophytic Streptomyces isolated from

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

Vol. 28

No. 1

2013

34

TANVIR et al.

Table 1. Morphological characteristics of the selected endophytic Streptomyces used in the study
Strain
RT6

Colony
size, mm
3

Colony shape
Circular

Margin
Undulate

Color of sub Color of aerial

strate
mycelium
Light brown

Light grey

Texture
Dry

Pigmentation
None

RT7

Circular

Undulate

Light brown

Dark grey

Dry

None

RT 10

Circular

Errose

Light brown

Light brown

Dry

None

RT11

Circular

Entire

Dark brown

Light brown

Dry

None

RT12

Circular

Entire

Dark brown

White

Dry

None

RT13

Circular

Entire

Dark brown

Grey

Dry

None

RT14

Circular

Entire

Light brown

Light pink

Dry

None

RT15

Irregular

Errose

Light yellow

White

Dry

None

RT18

Circular

Undulate

Brown

Light brown

Dry

None

RT34

Irregular

Undulate

Light brown

White

Dry

None

RT36

Circular

Entire

Brown

Grey

Dry

None

RT37

Circular

Entire

Brown

Light brown

Dry

None

RT38

Circular

Entire

Light brown

White

Dry

None

RT39

Circular

Entire

Brown

Light brown

Dry

None

RT40

Circular

Entire

Brown

Grey

Dry

None

RT41

Circular

Entire

Brown

Grey

Dry

None

RT43

Circular

Entire

Brown

Light grey

Dry

None

RT44

Circular

Entire

Light brown

Light brown

Dry

None

RT46

Circular

Errose

Black

Grey

Dry

None

RT47

Circular

Undulate

Brown

Light grey

Dry

None

RT48

Circular

Undulate

Brown

Grey

Dry

None

RT49

Circular

Entire

Light brown

Grey

Dry

None

RT50

Circular

Entire

Brown

Light Grey

Dry

None

RT51

Circular

Entire

Light brown

White

Dry

None

RT52

Circular

Entire

Brown

Light pink

Dry

None

RT53

Circular

Undulate

Dark brown

Light brown

Dry

None

RT54

Circular

Entire

Dark brown

Light brown

Dry

None

RT55

Circular

Entire

Dark brown

Light Grey

Dry

None

RT56

.3

Circular

Entire

Brown

LightGrey

Dry

None

RT57

Circular

Entire

Brown

Light Grey

Dry

None

RT58

Circular

Entire

Brown

Light Grey

Dry

None

RT59

Circular

Entire

Brown

White

Dry

None

RT60

Circular

Entire

Brown

White

Dry

None

RT61

Circular

Errose

Yellow

Light yellow

Dry

None

RT62

Circular

Entire

Dark brown

Light pink

Dry

None

RT63

Circular

Errose

Light Yellow

Yellow

Dry

None

RT64

Circular

Errose

Light Yellow

LightGrey

Dry

None

RT65

Circular

Undulate

Brown

Light brown

Dry

None

RT66

Circular

Entire

Brown

Grey

Dry

None

RT67

Circular

Entire

Brown

Grey

Dry

None

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

Vol. 28

No. 1

2013

SCREENING FOR TYPE I POLYKETIDE SYNTHASES GENES

35

Table 2. Melanin production and utilization of nine different sugars as carbon source of the selected endophytic Strepto
myces used in the study
Strain no.
RT6
RT7
RT10
RT13
RT14
RT18
RT36
RT37
RT38
RT39
RT40
RT41
RT43
RT44
RT46
RT47
RT49
RT50
RT53
RT54
RT55
RT56
RT57
RT58
RT59
RT60
RT61
RT63
RT64
RT65
RT67

Formation of
melanin

Glu

Fru

Raf

Rha

Ara

Man

Lac

Gal

Sue

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+

+
+
+
+
+
+
+
+
+

+
+

+
+

+
+
+
+

+
+

+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+

+
+

+
+
+

+
+
+
+
+
+
+
+

+
+
+
+
+

+
+
+
+
+
+

+
+

+
+

+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+

+
+
+
+
+
+
+
+
+

+
+
+

+
+
+
+
+
+
+

+
+
+

+
+
+
+
+

+
+
+
+
+

+
+
+
+
+

+
+

+
+

+
+
+
+
+
+
+
+
+
+

+
+
+

+
+
+

Note: Glu, Glucose; Fru, Fructose; Raf, Raffinose; Rha, Rhamnose, Ara, Arabinose; Man, Mannitol, Lac, Lactose; Gal, Galactose;
Suc, Sucrose.

P. hysterophorus has not been elucidated therefore we

screened 40 bioactive secondary metabolites producer
strains for the presence of PKS type I genes. For this
purpose six set of degenerate primers were prepared
specifically with high amino acid sequence identity for
the actinomycetes type I polyketide synthases. Step
down PCR amplification was done since this method
enables fast detection of PKS gene sequences inside
actinomycetes [11].
The PCR amplification with the primer set 1 and 4
revealed a band of ~300 bps for the PKS type I gene in

30 Streptomyces strains. The primer set 2 and set 3

exposed a band of ~320 bps in 25 and 11 strains respec
tively, whereas for primer set 5 and 7 a band of ~700 bps
in the 10 strains of endophytic Streptomyces was
detected. Majority of the screened isolates exhibited
the presence of PKS I gene amplification. PCR ampli
fication was obtained in the isolates RT13, RT43,
RT47, RT49, RT54, RT56, RT57, RT58, RT59,
RT61 and RT65 through all the six sets of primers
used (Table 6). It reveals that these endophytic Strep
tomyces contained a PKS I gene cluster responsible for

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

Vol. 28

No. 1

2013

36

TANVIR et al.

Table 3. Biochemical and physiological characterization of the selected endophytic Streptomyces used in the study
Strain no.
RT6
RT7
RT10
RT13
RT14
RT18
RT36
RT37
RT38
RT39
RT40
RT41
RT43
RT44
RT46
RT47
RT49
RT50
RT53
RT54
RT55
RT56
RT57
RT58
RT59
RT60
RT61
RT63
RT64
RT65
RT67

Formation of
organic acids

Utilization of
organic acids

Utilization of
oxalate

+
+

+
+
+
+
+
+
+
+
+

+
+
+

++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++

++
++
++
++
++
++

Hydrolysis of escu Hydrolysis of urea

lin and arbutin
and allantoin
+
+
+

+
+
++
+
++
+
+
+
++
++
+

+
+
++

++
++
+
+
++
++
+
+
+
+

Note: () negative, (+) moderate positive, (++) strong positive.

the production of the major bioactive secondary

metabolites. These results are supported by another
study on endophytic actinomyces isolated from three
medicinal plants in China and PKS genes was found to
be present in the majority of the isolates [14]. Another
study carried out on medicinal plants in Chinese for
ests also revealed PCR amplification prox. 0.5 g/wet
weight of mycelia was washed with 500 L of TE
buffer. The mycelia were collected by centrifugation
and resuspended in TE buffer with 20 L of 50 mg/mL
lysozyme and incubated at 37C for 24 hours. Then
50 L of 2% (w/v) SDS and 5 L of 20 mg/mL of pro
teinase K was added and incubated at 37C for 2
4 hours. After the second incubation 400 L of phe
nolchloroformisoamyl alcohol (25 : 24 : 1) was

added and centrifugation was done at 12000 rpm for

10 minutes. Supernatant was collected and transferred
to another tube, this step was repeated twice. Then 2
absolute ethanol was added and kept at 20C over
night. Centrifugation was done at 12000 rpm for
15 minutes and the pellet was washed with 70% etha
nol. The DNA was resuspended in 100 L TE buffer.
PKS type I gene amplification. PKSI gene ampli
fication was carried out using six set of degenerate
primers MAK1/MAK3, PO1/PO3 [11], PS11/PS12,
BKS254F/BKS960R, BKS1F/BKS1R and BKS2F/
BKS2R [9] by the step down PCR procedure (Table 5).
The 50 L reaction mixture was run using Fermentas
Dream Taq Green PCR Master Mix (2) (cat # K1081)

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

Vol. 28

No. 1

2013

SCREENING FOR TYPE I POLYKETIDE SYNTHASES GENES

37

Table 4. Results of 16S rRNA gene sequencing of the endophytic streptomycetes

Streptomyces strains

No. of nucleotides
sequenced (bp)

RT6
RT13
RT18
RT36
RT46
RT49
RT54
RT56
RT57
RT63
RT64
RT67

1425
1442
1444
1445
1445
1444
1445
1442
1445
1444
1443
1444

Streptomyces spp.
Streptomyces rochei
Streptomyces litmocidini
Strepomyces rochei
Streptomyces rochei
Streptomyces enissocaesili
Streptomyces djakartensis
Streptomyces olivaceus
Streptomyces spp.
Streptomyces plicatus
Streptomyces geysiriensis
Streptomyces spp.
Streptomyces vinaceusdrappus

% Homology GenBank accession no.

99%
99%
99%
99%
99%
99%
99%
99%
99%
99%
99%
99%

HQ909753
HQ909754
HQ909755
HQ909756
HQ909757
HQ909758
HQ909759
HQ909760
HQ909761
HQ909762
HQ909763
HQ909764

Table 5. Polyketide synthases type I (PKSI) primers used in the study

Primer sets

Sequence

References

MAK1

5' GACACSGCSTGYTCBTCGTCG 3'

Savic arid Vasiljevic, 2006

MAK3

5' CCGTTSGACGCRCCGTGGTTSAC 3'

Savic and Vasiljevic, 2006

PO1

5' GCNTGTMGNCTNTYYCCNGGNGG 3'

Savic and Vasiljevic, 2006

PO3

5' CTGTGSCGSACYAGBAGCAGC 3'

Savic and Vasiljevic, 2006

PS11

5' GGNACNCCNMANGGNGAMCC 3'

Lopanik et al., 2006

PS12

5' CGNYGGAANSGGTANGTNGG 3'

Lopanik et al., 2006

BKS254F

5' RMWYGGACCSCARCARC 3'

Lopanik et al., 2006

BKS960R

5' AKYTCGATBGGRTCRCCCAG 3'

Lopanik et al., 2006

BKS1F

5' GGTGCGGAGACGGGTGATTA 3'

Lopanik et al., 2006

BKS1R

5' CAACAAGAGCGGAGGAACAGGTA 3'

Lopanik et al., 2006

BKS2F

5' TTGGCACGTTGATCGAAGGTAAA 3'

Lopanik et al., 2006

BKS2R

5' TTGCCGAGTCGTTGTTAATAGTCT 3'

Lopanik et al., 2006

with 3 L template and 2 L forward and reverse prim

ers. The thermal cycler (Primus 96 Advanced Gradi
ent Peqlab, Germany) was programmed for 30 cycles
according to the following amplification profile: 30 sec
onds denaturation at 95C, 1 minute annealing at 77
53C, 1 minute extension at 72C where the annealing
temperature decreased 2C per cycle, followed by
30 seconds denaturation at 95C for 19 cycles, 1 minute
annealing at 51C, 1 minute elongation at 72C and final
extension step at 72C for 10 minutes [11].
RESULTS AND DISCUSSION
Streptomyces species live as saprophytes found gen
for PKSI and PKSII and NRPS biosynthetic sys
tems. They detected PKSII and NRPS sequences in

their Streptomyces isolates, however unlike the results

in our study the PKSI sequences were found to be less
abundant [8].
During the PCR screening however several isolates
RT11, RT12, RT15, RT34; RT48, RT51, RT52,
RT62 and RT66 revealed no gene amplification
through any of the six primers set which was also a
clear indication that these endophytic Streptomyces
did not contain the PKS I gene and therefore may not
be capable of producing any bioactive secondary
metabolite.
Our results clearly indicate that PKS type I genes
are widespread in the endophytic Streptomyces strains
isolated from P. hysterophorus L. The study suggests
that these endophytic Streptomyces may prove to be a
promising source of new bioactive agents.

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

Vol. 28

No. 1

2013

38

TANVIR et al.

Table 6. Screening for PKSI gene in selected endophytic Streptomyces isolates

Strain no.

Primer set no. 1: Primer set no. 2: Primer set no. 3:

MAK1, MAK3
PO1, PO3
PS11, PS12

Primer set no. 4:

Primer set no. 5: Primer set no. 6:
BKS254F,
BKS1F, BKS1R BKS2F, BKS2R
BKS960R

RT6

RT7

RT10

RT11

RT12

RT13

RT14

RT15

RT18

RT34

RT36

RT37

RT38

RT39

RT40

RT41

RT43

RT44

RT46

RT47

RT48

RT49

RT50

RT51

RT52

RT53

RT54

RT55

RTS6

RT57

RT58

RT59

RT60

RT61

RT62

RT63

RT64

RT65

RT66

RT67

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

Vol. 28

No. 1

2013

SCREENING FOR TYPE I POLYKETIDE SYNTHASES GENES

REFERENCES
1. Alam, M.T., Merlo, M.E., Takano, E., and Breitling, R.,
Mol. Phylogenet. Evol., 2010, vol. 54, pp. 763777.
2. BarriosLlerena, M.E., Burjaand, A.M., and Wright, P.C.,
J. Ind. Microbiol. Biotechnol., 2007, vol. 34, pp. 443456.
3. Gagunashvili, N., Davidsson, S.P., Jonsson, Z.O., et al.,
Mycol. Res., 2009, vol. 113, pp. 354363.
4. Germaine, K., Keogh, E., GarciaCabellos, G., et al.,
FEMS Microbiol. Ecol., 2004, vol. 48, pp. 109118.
5. Jung, W.S., Lee, S.K., Hong, J.S., et al., Appl. Micro
biol. Biotechnol., 2006, vol. 72, pp. 763769.
6. Komaki, H. and Harayama, S., Actinomycetologica,
2006, vol. 20, pp. 4248.
7. Kuster, E. and Williams, S.T., Nature, 1964, vol. 202,
pp. 928929.
8. Li, J., Zhao, G.Z., Chen, H.H., et al., Lett. Appl.
Microbiol., 2008, vol. 47, pp. 574580.

39

9. Lopanik, N.B., Targett, N.M., and Lindquist, N., Appl.

Environ. Microbiol., 2006, vol. 72, pp. 79417944.
10. Sajid, I., Shaaban, K.A., Yao, C.B.F., et al., J. Micro
biol. Biotechnol., 2009, vol. 25, pp. 601610.
11. Savic, M. and Vadiljevic, B., J. Ind. Microbiol. Biotech
nol., 2006, vol. 33, pp. 423430.
12. Shirling, E.B. and Gottlieb, D., Int. J. Syst. Bacteriol.,
1966, vol. 16, pp. 313340.
13. Varga, J., Rigo, K., Kocsube, S., et al., Res. Microbiol.,
2003, vol. 154, pp. 593600.
14. Wu, Y., Lu, C., Qian, X., et al., Curr. Microbiol., 2009,
vol. 59, pp. 475482.
15. Zhang, J., Lanen, S.G.V., Ju, J., et al., Proc. Natl. Acad.
Sci. U.S.A., 2008, vol. 105, pp. 14601465.
16. Zhao, X.Q., Jiao, W.C., Jiang, B., et al., J. Microbiol.
Biotechnol., 2009, vol. 25, pp. 859866.

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

Vol. 28

No. 1

2013