Scholarly article on topic 'Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4'

Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4 Academic research paper on "Industrial Biotechnology"

Share paper
Academic journal
OECD Field of science

Academic research paper on topic "Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4"

Int. J. Mol. Sci. 2014, 15, 12217-12230; doi:10.3390/ijms150712217


International Journal of

Molecular Sciences

ISSN 1422-0067


Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4

Feng-Hui Sun 12,3, Di Luo 12, Dan Shu 12, Juan Zhong 1,2,3 and Hong Tan 12*

1 Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, No. 9 Section 4, Renmin Nan Road, Chengdu 610041, China; E-Mails: (F.-H.S.); (D.L.); (D.S.); (J.Z.)

2 Environmental Microbiology Key Laboratory of Sichuan Province, No. 9 Section 4, Renmin Nan Road, Chengdu 610041, China

3 University of the Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China

* Author to whom correspondence should be addressed; E-Mail:; Tel./Fax: +86-28-8289-0409.

Received: 13 February 2014; in revised form: 21 March 2014 /Accepted: 24 March 2014 / Published: 9 July 2014

Abstract: To introduce DNA into Streptomyces noursei xinao-4, which produces xinaomycins, we explored an intergeneric conjugal transfer system. High efficiency of conjugation (8 x 10 exconjugants per recipient) was obtained when spores of S. noursei xinao-4 were heat-shocked at 50 °C for 10 min, mixed with Escherichia coli ET12567 (pUZ8002/pSET 152) in the ratio of 1:100, plated on 2CMY medium containing 40 mmol/L MgCl2, and incubated at 30 °C for 22 h. With this protocol, the plasmids pKC1139 and pSET152 were successfully transferred from E. coli ET12567 (pUZ8002) with different frequencies. Among all parameters, the ratio of donor to recipient cell number had the strongest effect on the transformation efficiency. In order to validate the above intergeneric conjugal transfer system, a glycosyltransferase gene was cloned and efficiently knocked out in S. noursei xinao-4 using pSG5-based plasmid pKC1139.

Keywords: Streptomyces noursei; xinaomycins; intergeneric conjugation; glycosyltransferase

1. Introduction

Streptomyces are soil-dwelling Gram-positive bacteria, and produce various bioactive molecules including half of microbial origin antibiotics employed in the field of medicine and agriculture [1,2]. Peptidyl nucleoside antibiotics are a group of secondary metabolites with similar chemical structures, which possess impressive antitumoral, antiviral, antibacterial, and antifungal activities [3,4]. The biosynthesis of peptidyl nucleoside antibiotics has been actively pursued due to their unique structures and clinically-potential applications recently. In the past few years, the biosynthetic gene clusters had been identified for nikkomycin [5], mildiomycin [3,6], gougerotin [7], blastididin S [8], puromycin [9], polyoxin [10], and streptothricin F [11]. Xinaomycins are a group of Streptomyces-derived peptidyl nucleoside antibiotics with potential antibacterial [12], antifungal [13], and antiviral [14,15] activities produced by S. noursei xinao-4 that has been isolated by our lab [16]. In order to improve xinaomycins production and get the highly pure xinaomycins, it is desirable to study the xinaomycins biosynthetic gene clusters. However, the fact that the genetic manipulation system of S. noursei xinao-4, such as gene cloning, transformation of vectors, and gene deletion has not been developed retards its molecular genetic studies. The present study aimed at the development of an efficient transformation system for the S. noursei xinao-4 in order to carry out the genetic manipulation of this strain.

There are three genetic transformation systems available that have been used to transfer foreign plasmids into Streptomyces. The protoplast transformation and electroporation techniques have been widely used [17,18]. Unfortunately, these two methods are relatively inefficient mainly because of the strain-specific conditions for protoplast formation and regeneration, and also the presence of different restriction-modification systems in each strain. Intergeneric conjugation between Escherichia coli and Streptomyces which was firstly reported in 1989 [19], was proved to be a reliable method. Especially the employment of methylation-deficient E. coli as donor and the construction of various E. coli shuttle vectors [20] improved this method, so that it was used widely in the Streptomyces [1,21-26]. Although transformation systems for several Streptomyces strains have been developed since 1989 [1,21-26], there is still no universal protocol applicable to all Streptomyces strains. We have carried out an intergeneric conjugation according to the standard procedure described by Kieser et al. [22]. Unfortunately, few exconjugants harboring integrative plasmid pSET152 were received at low efficiency (about 10-6), and no deletion mutants (conducted by suicide vector pOJ260) was obtained.

In this study, we report a reliable genetic system for S. noursei xinao-4 by optimization of transformation conditions including conjugal medium, heat-shock, and ratio of donor to recipient cell number, as well as incubation time of the conjugation plates. The conjugation efficiency of integrative and self-replicative plasmids and the effect of these two plasmids on xinaomycins production were also evaluated. A glycosyltransferase (glt) gene was successfully disrupted based on the temperature sensitive plasmid pKC1139 with the established transformation system. We developed an efficient intergeneric conjugation procedure to introduce the plasmid DNA into S. noursei xinao-4 that may form the basis of gene functional analysis.

2. Results and Discussion

2.1. Antibiotic Tolerance of S. noursei Xinao-4

In order to establish an efficient transformation system, we initially studied the antibiotic tolerance of S. noursei xinao-4. The results showed that the organism was sensitive to thiostrepton (at concentration >6.25 |ig/mL), neomycin (>12.5 |ig/mL), kanamycin (>25 |ig/mL), and apramycin (>12.5 |ig/mL). The S. noursei xinao-4 shows some background growth on YMS media including <25 |ig/mL spectinomycin. It is sensitive to apramycin and thiostrepton at concentrations >12.5 |ig/mL and 6.25 |ig/mL respectively. Thus, these two antibiotics are favorable for selection of exconjugants.

E. coli ET12567 (pUZ8002) containing pSET152 was used as the donor in the following optimization of intergeneric conjugation. Several parameters that might affect the efficiency of conjugation were evaluated, e.g., mating solid media, heat-shock, ratio of donor to recipient cell number, and incubation time of the conjugation plates.

2.2. Optimization of Solid Media for Exconjugants Regeneration

Previous studies showed that different conjugal medium played a significant role for the conjugal efficiency and the appropriate conjugal medium varied between different Streptomyces strains [1,24]. To select the appropriate medium for intergeneric conjugation, five agar media (MS, ISP4, 2CMY, YMS and TSA) containing 10 mmol/L MgCl2 were assessed as conjugative medium. Among them, MS, ISP4 and 2CMY has been used as conjugal medium for several Streptomyces [1,21,25-28]; TSA is usually applied in the growth of Streptomyces species [1,22]; and YMS is the suitable medium for the growth and spore formation of S. noursei xinao-4. S. noursei xinao-4 grow and sporulate fast within 7 days on YMS agar at 28 °C. The results (data not shown) showed that TSA and YMS were not suitable for conjugation because E. coli outgrew over S. noursei xinao-4 during overnight incubation, with few exconjugants (about 1 x 10-6) on these two media. When compared to the conjugation frequency of MS medium and ISP4 medium, the conjugation frequency of 2CMY medium increased 1.8 and 1.3 times, respectively, reaching (6.04 ± 0.68) x 10-5. Thus, 2CMY medium was proved to be the most appropriate medium for intergeneric conjugation.

Generally, MgCl2 has been added to the conjugation medium to improve the efficiency of conjugation [22,23]; however, optimal concentration of MgCl2 differed between Streptomyces strains. In our study, the conjugation medium 2CMY was further optimized with a supplement of 0, 10, 20, 30, 40, 50 mM of MgCl2, respectively. As the results shown (Figure 1), conjugation efficiency was further increased, in the presence of higher concentrations of MgCl2. The highest conjugation efficiency was achieved in the presence of 40 mM MgCl2. The conjugation efficiency decreased dramatically, when the concentration was higher than 40 mM MgCl2, e.g., 50 mM. Our results revealed that 40 mM MgCl2 appeared to be the optimal concentration for conjugation of S. noursei xinao-4.

Figure 1. Effect of the concentration of MgCl2 on conjugation efficiency with E. coli ET12567 (pUZ8002/pSET152) on 2CMY agar medium.

2.3. Effect of Heat-Shock on the Conjugation Efficiency

Heat-shock of the Streptomyces spores has been generally recommended before mating with E. coli donors [19,21,22]. To determine the optimal temperature for heat treatment, we evaluated the effect of heat treatment with different temperature on the spore viability and conjugation efficiency. As shown in Table 1, we found that the efficiency of conjugation increased along with temperature rising until reaching 50 °C, but decreased rapidly above 50 °C. The highest efficiency was achieved using a 50 °C heat shock, and was about 10 times that obtained at room temperature. As expected, the spore viability decreased as the temperature of heat-treatment increased, and the spores lost their viability quickly when they were incubated at 55 °C for 10 min. Apparently, the conjugation efficiency was not consistent with spore viability, indicating that the effect of heat-treatment on conjugation efficiency might be attributed to the temporal reduction of the restriction enzyme activities, other than increasing the viability of spores [29,30].

Table 1. Effect of heat treatment of S. noursei xinao-4 spores on spore viability and conjugation efficiency with E. coli ET12567 (pUZ8002/pSET152) on 2CMY agar medium containing 10 mM MgCl2.

Temperature (°C) Viability (%) Conjugation Frequency

Room temperature 100 (4.87 ± 0.31) x 10-5

40 95.5 (8.0 ± 0.56) x 10-5

45 69.87 (1.96 ± 0.29) x 10-4

50 56.12 (3.47 ± 0.21) x 10-4

55 17.97 (7.1 ± 0.45) x 10-5

Incubation of heat-shocked spores prior to mixing with E. coli was recommended for many Streptomyces strains [23,31]. Therefore, the effect of incubation time of heat-shocked spores on the

conjugation efficiency was also investigated. As shown in Figure 2, the longer incubation of heat-shocked spores was, the fewer exconjugants were received. Thus, we treated the S. noursei xinao-4 spores at 50 °C for 10 min without further cultivation for conjugation.

Figure 2. Effect of incubation of heat-treated spores on conjugation efficiency with E. coli ET12567 (pUZ8002/pSET152) on 2CMY agar medium containing 10 mM MgCh.

<l) 3.5-n

c Q 2.5

ü ^ t £ a) CD

O E Ä 1=


8 0.5-

Incubation time (h)

2.4. Effect of Donor-to-Recipient Ratio on Conjugation Efficiency

The ratio of donor cell number to recipient spore number was a crucial parameter in the intergeneric conjugation of Streptomyces. One mL of S. noursei spores at a concentration of 10 per mL, heat-treated

at 50 °C for 10 min and a range of E. coli ET12567 exponential cells (10-10 ) were mixed, and the conjugation efficiency was evaluated. As shown in Table 2, few exconjugants were obtained

when the number of donor cells was less than 10 . When the number of the donor cells was up to 10 ,

the conjugation efficiency reached the highest efficiency of (2.3 ± 0.24) x 10 , which is 34 times greater than that obtained with 10 donor cells. In contrast with the results of Streptomyces coelicolor [21] and Streptomyces nodosus [25], where the donor-to-recipient ratio did not affect the conjugal efficiency, our results indicated that the maximum efficiency was received with a certain donor-to-recipient ratio of 100:1. Similar results had been observed in Kitasatospora setae [32], Streptomyces lincolnensis [24], and Streptomyces diastatochromogenes 1628 [31], Streptomyces natalensis [33] in which a certain ratio of donor to recipient cell number was required to achieve highest frequency.

Table 2. Effect of donor-to-recipient on conjugation efficiency with E. coli ET12567 (pUZ8002/pSET152) on 2CMY agar medium containing 10 mM MgCh.

Donor Numbers (x107) Conjugation Frequency

1 (6.59 ± 0.29) x 10-5

10 (5.5 ± 0.27) x 10-4

100 (2.3± 0.24) x 10-3

1000 (4.4 ± 0.2) x 10-4

2.5. The Effect of Incubation Time of Mix-Culture on the Conjugation Efficiency

The intergeneric conjugation, especially, the plasmid transfer, takes place during the incubation of the mixed culture of E. coli and Streptomyces, stressing the importance of incubation time of mix-culture of these two strains. If the incubation time is too short, the conjugation cannot be accomplished, resulting in lower conjugation efficiency. On the contrary, the longer the incubation time, the greater is the probability of false positives. As shown in Figure 3, more exconjugants were received with an incubation time prolonged from 12 to 22 h. There was little difference in conjugation efficiency among 18, 20 and 22 h incubation time. Nevertheless, when the incubation time was extended to 24 h, there were many false positive exconjugants on the plate, which hampered the selection of the positive exconjugants. Our results indicated that the best incubation time of mix-culture plates was 18-22 h.

Figure 3. Effect of incubation time of mixed culture on conjugation efficiency with E. coli ET12567 (pUZ8002/pSET152) on 2CMY agar medium containing 10 mM MgCl2.

b - 3 5-

El ¡¡3

c 9 o E

~ § 2 5-

o g> 2.0-

Incubation time (h)


2.6. Conjugal Transfers of Integrated and Self-Replicative Plasmid into S. noursei Xinao-4

In order to assess the availability of the present intergeneric conjugal transfer system, two common Streptomyces plasmids, pKC1139 and pSET152 were transferred into S. noursei xinao-4. Both plasmids used in these transformation experiments contain the origin of transfer, oriT from plasmid RP4 allowing mobilization of the single strand during conjugation from E. coli ET12567 into Streptomyces. The plasmids were successfully transferred into S. noursei xinao-4 albeit with different frequencies. The frequency of plasmid pKC1139 was lower than that of integrative plasmid pSET152. The conjugation frequency was up to (8.0 ± 0.14) x 10-3 per recipient for S. noursei xinao-4 under optimal conditions with integrative plasmid pSET152, while the frequency of plasmid pKC1139 was (3.5 ± 0.24) x 10-5.

The PCR experiments confirmed that all randomly chosen exconjugants displayed a 519 bp band of the apramycin resistance gene, demonstrating the presence of the plasmid (data not shown). The exconjugants were stable after five successive sub-cultivations, indicating that this intergeneric

conjugation strategy can be widely applied for genetic research in the future. All exconjugants did not differ from the wild-type strain, S. noursei xinao-4, in their ability to produce xinaomycins. Besides that, we did not observe any changes in the sporulation or morphology of the exconjugants on YMS plates when compared to the wild-type strain (data not shown).

2.7. Validation of Conjugation System: A Case Study

Suicide vectors and pSG5-based temperature-sensitive plasmids were widely used for gene knock-out in Streptomyces. In order to compare these two kinds of plasmids for deletion in S. noursei xinao-4 and validate the established intergeneric transformation system, a 3230 bp fragment of putative glycosyltransferase gene (glt) was cloned from its genomic DNA. It was reported that glycosyltransferase can transfer glycosyl into various acceptors, such as oligosaccharides, lipids, proteins, nucleic acids and innumerous natural products [34,35], such as in the biosynthesis of nucleoside antibiotics, cytosinpeptidemycin Streptomyces ahygroscopicus [36] and amicetin in Streptomyces vinaceusdrappus [35,37]. This enzyme may be involved in xinaomycins biosynthesis. We cloned a glycosyltransferase gene (glt) from the genomic DNA of S. noursei xinao-4 by degenerate primers. The obtained fragment shows high sequence similarity with many glycosyltransferase genes from other Streptomyces. The two vectors pOJ2601 and pKC1139-1 for glt knock-out were constructed as above.

About one single crossover exconjugant was obtained per 1010 spores using suicide vector pOJ2601. Unfortunately, we failed to receive any double-crossover mutants by pOJ2601 directly. One of the single-crossover exconjugants was allowed three rounds of sporulation on YMS agar without antibiotic selection and its progeny were screened for double-crossover mutants. About one thousand colonies were screened, three of which were confirmed to be double-crossover mutants. The low efficiency of double-crossover mutants with suicide vector may be attributed to the short homologous arm (about 1 kb) present in plasmid pOJ2601.

About 103 transformants were obtained using the plasmid pKC1139-1, twelve exconjugants of which were verified by PCR and further incubated for sporulation at 30 °C. The harvested spores were spread on YMS plate containing thiostrepton and incubated at 37 °C for 7 days to eliminate the free plasmids from the host cells. The spores were collected and spread on YMS agar containing thiostrepton or apramycin to screen the double-crossover mutants. Five hundred colonies were chosen, and nineteen double-crossover mutants of which were obtained. Three mutants were randomly selected and verified by PCR. As shown in Figure 4, mutants have 571 bp deletions of glt gene and showed a 1509 bp fragment with the gltp7 and gltp8 as the primer.

When compared to wild-type strain, S. noursei xinao-4, the glt mutants exhibited no visible change on xinaomycins synthesis and morphogenesis by shake flask fermentation in BL medium, suggesting that deletion of glt had no effect on xinaomycins production under the condition we tested. Nevertheless, the glt gene was disrupted successfully by above protocol, and 22 deletion mutants were obtained with these two plasmids. Apparently, temperature-sensitive plasmids had a greater chance to acquire deletion mutants, which provided a practical tool for further genetic manipulation of S. noursei xinao-4.

Figure 4. Inactivation of glycosyltranferase gene by gene deletion. (A) Construction of deletion mutants of glt; and (B) Verification of the double crossover mutants by PCR from wild-type (1022 bp) and mutant strain (1509 bp). Lane 1: wild-type strain; lane 2: mutant strain; M: marker.

3. Experimental Section

3.1. Strains and Plasmids

E. coli DH5a was used as general host for cloning and plasmid reproduction [38]. Methylation-deficient E. coli ET12567 (dam-13::Tn 9, dcm-6, hsdM, hsdS) [39], harboring plasmid pUZ8002 [40] was used as the donor for conjugation experiments. S. noursei xinao-4, the xinaomycins producer, was maintained in our lab and used as recipient in intergeneric conjugation. The replicative vector pKC1139 [20] contains pSG5 origins of replication and intergrative vector pSET152 [20] contains int-attp fragments of actinophages ^C31, were used as conjugative vectors to transfer from E. coli ET12567 to S. noursei xinao-4. pEASY-Blunt Zero (TransGen Biotech, Beijing, China) vector was used for cloning of the PCR products. The conjugative plasmids p0J260 [20] and pKC1139 were used to construct the deletion vectors. The plasmid pWHM3 [41] served as the template to amplify the thiostrepton resistance gene. The glt deletion vectors p0J2601 and pKC1139-1 were constructed as described below. Bacillus cereus was used as indicator for bioassay of xinaomycins.

3.2. Media and Culture Condition

E. coli strains were grown in Luria-Bertani (LB) medium supplemented with appropriate antibiotics at 200 rpm in a shaking incubator at 37 or 30 °C as required [42]. S. noursei xinao-4 was routinely grown on YMS agar at 28 °C. The spores of S. noursei xinao-4 were achieved on YMS medium at 30 °C for 7 days, and then scraped and used as recipients. Mycelia for DNA extraction were grown in TSB broth containing 1% glycine (w/v) at 28 °C for 2 days [22]. MS [43], ISP4 [44], 2CMY [45], Tryptone soya agar (TSA) [22] and YMS [46] plates were used as the conjugative media. Apramycin (50 |ig/mL), ampicillin (50 |ig/mL), chloramphenicol (25 |ig/mL), kanamycin (50 |ig/mL), thiostrepton (25 |ig/mL) and nalidixic acid (50 |ig/mL) was added as required.

3.3. Construction of Plasmids for Gene Deletion

The constructions of vectors for gene inactivation were carried out according to Shevchuk et al. [47]. A 1058 bp fragment designated as tsr, including the coding region of thiostrepton resistance gene and its promoter sequence was amplified from the plasmid pWHM3 with tsrF (5'-TGATCAAGGCGAA TACTTCAT-3') and tsrR (5'-TCATCACTGACGAATCGAGGT-3'). A 1216 bp fragment designated gltl, encompassing the region upstream of glt gene and some of its encoding region, was amplified from the S. noursei xinao-4 genomic DNA with primers gltpl (5'-CTCGCCCATGTTCCCGTAC-3') and gltp2 (5'-ATGAAGTATTCGCCTTGATCAACCGCCGTTCTTCTCGTGCC-3'). A 1236 bp fragment designated glt2, encompassing some of glt gene encoding region and the region downstream of this gene, was amplified with primers gltp3 (5'-ACCTCGATTCGTCAGTGATGATCTTCCTGG CCTCCACCCTCTA-3') and gltp4 (5'-GGCATCCGGTTGCTGTTC-3') (the sequence overlapping the tsr fragment is underlined). The obtained three fragments were ligated together according to the long multiple fusion (LMF) method [47]. The PCR reactions (50 |L) contained 0.5 U KOD-Plus-Neo DNA Polymerase (Toyobo, Shanghai, China), 5 |L 10* PCR Buffer for KOD-Plus-Neo, 1 mM of MgCl2, 200 ng of template DNA, 200 | M of each dNTP, 0.2 | M of each primer, and 2.5 | L of DMSO. PCR was performed as follows: after initial denaturation at 94 °C for 5 min, 30 cycles PCR reaction were performed at 94 °C for 30 s, 62 °C for 30 s and 68 °C for 1 min.

A 3409 bp fragment including the 1141 bp upstream homologous arm, the 1058 bp thiostrepton resistance cassette, and the 1210 bp downstream homologous arm, was obtained with the primers gltp5 (5'-CGCGGATCCGGGCAGTTCCTCGATGTGGGTG-3') (with a BamHI site, underlined) and gltp6 (5'-CCCAAGCTTGCTGTTCTGGTACGGCGTCAACC-3') (with a HindIII site, underlined). The obtained 3409 bp fragment was cloned to pEASY-Blunt Zero (TransGen Biotech, Beijing, China), and then digested with BamHI and HindIII. The resulting fragment was ligated to p0J260 or pKC1139, which had been cut with BamHI and HindIII endonuclease, yielding the glt deletion vector p0J2601 and pKC1139-1.

3.4. Intergeneric Conjugation Procedure and Analysis of Exconjugants

Intergeneric conjugation between E. coli and S. noursei xinao-4 was carried out as described by Kiser et al. [22] with some modification. A culture of the donor E. coli ET12567 (pUZ8002) containing conjugative plasmid was grown to an 0D600 of 0.4-0.6 with the appropriate antibiotics. To remove the antibiotics, the cells were collected and washed twice with LB, and then suspended in 0.1 volume of LB medium. S. noursei xinao-4 spores were collected with 2* YT broth after growth for seven days on YMS medium at 30 °C. Aliquots of fresh S. noursei xinao-4 spores were collected, washed twice and suspended in 2* YT broth at a concentration of 107 per mL. The S. noursei xinao-4 spores were heated at 40-50 °C for 10 min, incubated at 37 °C for 0-8 h and served as recipients. Donors and recipients were mixed and spread on MS, ISP4, 2CMY, TSB and YMS plates and grown for 12-24 h at 30 °C. The effect of MgCl2 concentration (0-50 mM) on conjugal efficiency was also evaluated. After incubation, the plates were covered with 1 mL water containing 0.625 mg nalidixic acid and 0.625 mg apramycin or thiostrepton as required, and incubated at 30 °C for 3-7 days, until the exconjugants appeared.

3.5. Analysis of Exconjugants

Total DNA of exconjugants was prepared according to the method of Kieser et al. [22]. A 519 bp fragment was amplified by PCR with the specific primers AAC-F (5'-TGGGCCACTTGGACTGAT-3') and AAC-R (5'-CCGACTGGACCTTCCTTCTG-3') within the apramycin resistance gene, demonstrating the presence of vector pSET152 or pKC1139. The knock-out mutants were verified by PCR with the specific primers gltp7 (5'-CGGAATGGGTACGTGATGG-3') and gltp8 (5'-GCCGAGATGGTGGGA AATG-3') within the homologous arms. The double crossover mutants revealed a 1509 bp fragment, differing from the 1022 bp fragment found in the wild type strain. The PCR reactions (50 цЬ) contained 2.5 U Easy Taq DNA Polymerase (TransGen Biotech, Beijing, China), 5 цЬ 10* Easy Taq buffer, 200 ng of template DNA, 250 цМ of each dNTP, 0.2 цМ of each primer, and 2.5 цЬ of DMSO. PCR was performed as follows: after initial denaturation at 94 °C for 5 min, 30 cycles PCR reaction were performed at 94 °C for 30 s, 62 °C for 30 s and 72 °C for 1 min.

3.6. Fermentations and Bioassay of Xinaomycins

Fermentation was carried out in 250 mL Erlenmeyer flasks containing BL medium (containing glucose 6.0 g/L, corn powder 6.0 g/L, yeast powder 0.5 g/L, soybean 9.0 g/L, KNO3 1.0 g/L, NaCl 3.0 g/L, (NH4)2SO4 2.0 g/L, CaCO3 2.0 g/L, and MgSO4'7H2O 0.5 g/L, pH 7.2). The seed culture was cultivated in 40 mL BL medium under 150 rpm, at 28 °C for 36 h. The fermentation culture (100 mL BL medium) was inoculated with 5 mL seed culture, and then incubated at 28 °C, under 150 rpm for 2 days. The fermentation broth was collected and centrifuged at 6000 rpm for 10 min. The liquid supernatant was collected and used to detect the xinaomycins [16].

The bioassay of xinaomycins was carried out with SC medium (peptone 1.5 g/L, beef extract 1.5 g/L, Na2HPO4 0.05 g/L, agar 18 g/L) according to the cup-plate method, containing Bacillus cereus, a strain that is sensitive to xinaomycins. 30 цL of fermentation broth were added to the oxford cup, and were then incubated for 17 h at 30 °C, and screened visually for growth-inhibition zones [12].

4. Conclusions

In conclusion, an efficient intergeneric conjugation system of transferring plasmids from E. coli to

5. noursei xinao-4 was developed. Compared to the previous studies, in which the transformation efficiency is below 10 3 [1,26,31,33,48], our study showed high transformation efficiency. The availability of this protocol was confirmed by the 22 double-crossover mutants of glycosyltranferase gene. This procedure lays a good foundation for DNA transformation and mutational analysis of this industrially important strain. Also, it becomes a useful tool for introducing deletions and alterations of the genes responsible for xinaomycins biosynthesis, exploring its natural biosynthetic pathway, understanding the regulation of xinaomycins in more details, and subsequently producing new hybrid derivates.


We thank Huarong Tan (Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) for providing plasmids pKC1139 and pSET152. We thank Weiying Liu (China Pharmaceutical

University, Nanjing, China) for providing E. coli ET12567 (pUZ8002), and Yanan Song for providing plasmid pOJ260 and pWHM3 (Nanjing University, Nanjing, China). This work is supported by the National High Technology Research, Development Program of China (2011AA10A205) and the Keynote Program of the Chinese Academy of Sciences (KSZD-EW-Z-021-3-1).

Author Contributions

F.H. Sun contributed to the laboratory work, data analyses and manuscript writing; D. Luo, D. Shu and J. Zhong conceived the study and commented the manuscript; H. Tan conceived and designed the study. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest. References

1. Phornphisutthimas, S.; Sudtachat, N.; Bunyoo, C.; Chotewutmontri, P.; Panijpan, B.; Thamchaipenet, A. Development of an intergeneric conjugal transfer system for rimocidin-producing Streptomyces rimosus. Lett. Appl. Microbiol. 2010, 50, 530-536.

2. Hopwood, D.A. Forty years of genetics with Streptomyces: From in vivo through in vitro to in silico. Microbiology 1999, 145, 2183-2202.

3. Li, L.; Xu, Z.; Xu, X.; Wu, J.; Zhang, Y.; He, X.; Zabriskie, T.M.; Deng, Z. The mildiomycin biosynthesis: Initial steps for sequential generation of 5-hydroxymethylcytidine 5'-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119. ChemBioChem 2008, 9, 1286-1294.

4. Isono, K. Nucleoside antibiotics: Structure, biological activity, and biosynthesis. J. Antibiot. 1988, 41, 1711-1739.

5. Bormann, C.; Mohrle, V.; Bruntner, C. Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae TU901 in Streptomyces lividans. J. Bacteriol. 1996, 178, 1216-1218.

6. Wu, J.; Li, L.; Deng, Z.; Zabriskie, T.M.; He, X. Analysis of the mildiomycin biosynthesis gene cluster in Streptoverticillum rimofaciens ZJU5119 and characterization of MilC, a hydroxymethylcytosyl-glucuronic acid synthase. ChemBioChem 2012, 13, 1613-1621.

7. Niu, G.; Li, L.; Wei, J.; Tan, H. Cloning, heterologous expression, and characterization of the gene cluster required for gougerotin biosynthesis. Chem. Biol. 2013, 20, 34-44.

8. Cone, M.C.; Yin, X.; Grochowski, L.L.; Parker, M.R.; Zabriskie, T.M. The blasticidin S biosynthesis gene cluster from Streptomyces griseochromogenes: Sequence analysis, organization, and initial characterization. ChemBioChem 2003, 4, 821-828.

9. Tercero, J.A.; Espinosa, J.C.; Lacalle, R.A.; Jiménez, A. The biosynthetic pathway of the aminonucleoside antibiotic puromycin, as deduced from the molecular analysis of the pur cluster of Streptomyces alboniger. J. Biol. Chem. 1996, 271, 1579-1590.

10. Chen, W.; Huang, T.; He, X.; Meng, Q.; You, D.; Bai, L.; Li, J.; Wu, M.; Li, R.; Xie, Z. Characterization of the polyoxin biosynthetic gene cluster from Streptomyces cacaoi and engineered production of polyoxin H. J. Biol. Chem. 2009, 284, 10627-10638.

11. Fernandez-Moreno, M.A.; Vallin, C.; Malpartida, F. Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation. J. Bacteriol. 1997, 179, 6929-6936.

12. Ning, T.B.; Zhang, T.; Zhou, J.Y.; Yang, J.; Hong, T. Extraction methods and physicochemical properties of fermentation broth of xinaomycin. Hunan Agric. Sci. 2011, 13, 23-25.

13. Gu, G.J.; Cui, L.K.; Wang, S.H.; Li, F.X.; Lin, X.M. Toxicity test of xinaomycin to seven plant pathogenic fungi in laboratory. Agrochemicals 2013, 52, 293-294.

14. Li, F.G.; Yang, F.; Feng, Z.Q.; Lu, Q. Field efficacy of 4% xinaomycin AS on tomato mosaix virus disease. J. Henan Agric. Sci. 2013, 42, 86-88.

15. Feng, Z.Q.; Lu, Q. Field efficacy of new bio-fungicide xinaomycin on tobacco mosaic virus. Mod. Agrochem. 2011, 10, 50-52.

16. Zhou, J.Y.; Hong, T.; Juan, Z.; Li, Z.D.; Yang, J.; Xiao, L.; Liu, D. Antimicrobial Compound and Preparation Method and Application. CN Patent 200910060121.4, 9 February 2011.

17. Matsushima, P.; Baltz, R.H. Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts. J. Bacteriol. 1985, 163, 180-185.

18. Voeykova, T.; Emelyanova, L.; Tabakov, V.; Mkrtumyan, N. Transfer of plasmid pTO1 from Escherichia coli to various representatives of the order Actinomycetales by intergeneric conjugation. FEMSMicrobiol. Lett. 1998, 162, 47-52.

19. Mazodier, P.; Petter, R.; Thompson, C. Intergeneric conjugation between Escherichia coli and Streptomyces species. J. Bacteriol. 1989, 171, 3583-3585.

20. Bierman, M.; Logan, R.; O'Brien, K.; Seno, E.T.; Nagaraja Rao, R.; Schoner, B.E. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 1992, 116, 43-49.

21. Flett, F.; Mersinias, V.; Smith, C.P. High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting Streptomycetes. FEMS Microbiol. Lett. 1997, 155, 223-229.

22. Kieser, T.; Bibb, M.J.; Chater, K.F.; Butter, M.J.; Hopwood, D.A. Practical Streptomyces Genetics: A Laboratory Manual; John Innes Foundation: Norwich, UK, 2000.

23. Gust, B.; Challis, G.L.; Fowler, K.; Kieser, T.; Chater, K.F. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. USA 2003, 100, 1541-1546.

24. Du, L.; Liu, R.-H.; Ying, L.; Zhao, G.-R. An efficient intergeneric conjugation of DNA from Escherichia coli to mycelia of the lincomycin-producer Streptomyces lincolnensis. Int. J. Mol. Sci. 2012, 13, 4797-4806.

25. Nikodinovic, J.; Barrow, K.D.; Chuck, J.-A. High frequency transformation of the amphotericin-producing bacterium Streptomyces nodosus. J. Microbiol. Methods 2003, 55, 273-277.

26. Guan, D.; Pettis, G. Intergeneric conjugal gene transfer from Escherichia coli to the sweet potato pathogen Streptomyces ipomoeae. Lett. Appl. Microbiol. 2009, 49, 67-72.

27. Jin, Z.; Jin, X.; Jin, Q. Conjugal transferring of resistance gene ptr for improvement of pristinamycin-producing Streptomyces pristinaespiralis. Appl. Biochem. Biotechnol. 2010, 160, 1853-1864.

28. Park, H.Y.; Jang, B.Y.; Hwang, Y.I.; Choi, S.U. Construction of intergeneric conjugal transfer for molecular genetic studies of Streptomyces acidiscabies producing thaxtomin. J. Korean Soc. Appl. Biol. Chem. 2012, 55, 265-269.

29. Bailey, C.R.; Winstanley, D.J. Inhibition of restriction in Streptomyces clavuligerus by heat treatment. J. Gen. Microbiol. 1986, 132, 2945-2947.

30. Engel, P. Plasmid transformation of Streptomyces tendae after heat attenuation of restriction. Appl. Environ. Microbiol. 1987, 53, 1-3.

31. Ma, Z.; Liu, J.; Bechthold, A.; Tao, L.; Shentu, X.; Bian, Y.; Yu, X. Development of intergeneric conjugal gene transfer system in Streptomyces diastatochromogenes 1628 and its application for improvement of toyocamycin production. Curr. Microbiol. 2013, 68, 180-185.

32. Choi, S.U.; Lee, C.K.; Hwang, Y.I.; Kinoshita, H.; Nihira, T. Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Kitasatospora setae, a bafilomycin b1 producer. Arch. Microbiol. 2004, 181, 294-298.

33. Enriquez, L.L.; Mendes, M.V.; Anton, N.; Tunca, S.; Guerra, S.M.; Martin, J.F.; Aparicio, J.F. An efficient gene transfer system for the pimaricin producer Streptomyces natalensis. FEMSMicrobiol. Lett. 2006, 257, 312-318.

34. Choi, S.H.; Kim, H.S.; Yoon, Y.J.; Kim, D.M.; Lee, E.Y. Glycosyltransferase and its application to glycodiversification of natural products. J. Ind. Eng. Chem. 2012, 18, 1208-1212.

35. Chen, R.; Zhang, H.; Zhang, G.; Li, S.; Zhang, G.; Zhu, Y.; Liu, J.; Zhang, C. Characterizing amosamine biosynthesis in amicetin reveals AmiG as a reversible retaining glycosyltransferase. J. Am. Chem. Soc. 2013, 135, 12152-12155.

36. Wei, Y.Y. Gene Transformation of Streptomyces ahygroscopicus, Cloning and Function of Cytosinpeptidemycin Biosynthetic Genes. Ph.D. Thesis, Shenyang University, Shenyang, China, 15 June 2009.

37. Zhang, G.; Zhang, H.; Li, S.; Xiao, J.; Zhang, G.; Zhu, Y.; Niu, S.; Ju, J.; Zhang, C. Characterization of the amicetin biosynthesis gene cluster from Streptomyces vinaceusdrappus NRRL 2363 implicates two alternative strategies for amide bond formation. Appl. Environ. Microbiol. 2012, 78, 2393-2401.

38. Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 1983, 166, 557-580.

39. MacNeil, D.J.; Gewain, K.M.; Ruby, C.L.; Dezeny, G.; Gibbons, P.H.; MacNeil, T. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 1992, 111, 61-68.

40. Paget, M.S.; Chamberlin, L.; Atrih, A.; Foster, S.J.; Buttner, M.J. Evidence that the extracytoplasmic function sigma factor sigma is required for normal cell wall structure in Streptomyces coelicolor A3(2). J. Bacteriol. 1999, 181, 204-211.

41. Vara, J.; Lewandowska-Skarbek, M.; Wang, Y.-G.; Donadio, S.; Hutchinson, C. Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus). J. Bacteriol. 1989, 171, 5872-5881.

42. Sambrook, J.; Russell, D. Molecular Cloning: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: New York, NY, USA, 2001.

43. Hobbs, G.; Frazer, C.M.; Gardner, D.C.; Cullum, J.A.; Oliver, S.G. Dispersed growth of Streptomyces in liquid culture. Appl. Microbiol. Biotechnol. 1989, 31, 272-277.

44. Shirling, E.B.; Gottlieb, D. Method for characterization of Streptomyces species. Int. J. Syst. Evolut. Microbiol. 1966, 16, 313-340.

45. Xiao, Y.; Li, S.M.; Ma, L.; Zhang, G.Y.; Ju, J.H.; Zhang, C.S. Genetic manipulation system for tiacumicin producer Dactylosporangium aurantiacum NRRL 18085. Acta Microbiol. Sin. 2010, 50, 1014-1022.

46. Ikeda, H.; Kotaki, H.; Omura, S. Genetic studies of avermectin biosynthesis in Streptomyces avermitilis. J. Bacteriol. 1987, 169, 5615-5621.

47. Shevchuk, N.A.; Bryksin, A.V.; Nusinovich, Y.A.; Cabello, F.C.; Sutherland, M.; Ladisch, S. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucleic Acids Res. 2004, 32, e19.

48. Sioud, S.; Aigle, B.; Karray-Rebai, I.; Smaoui, S.; Bejar, S.; Mellouli, L. Integrative gene cloning and expression system for Streptomyces sp. Us 24 and Streptomyces sp. Tn 58 bioactive molecule producing strains. Biol. Med. Res. Int. 2009, 2009, 464986:1-464986:10.

© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article

distributed under the terms and conditions of the Creative Commons Attribution license


Copyright of International Journal of Molecular Sciences is the property of MDPI Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.