Environment Health Techniques Characterization of plasmid pXL100 and construction of a shuttle vector
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Research Paper Characterization of plasmid pXL100 from Amycolatopsis orientalis HCCB10007 and construction of a shuttle vector Li Xu1,2, Yanmei Li2, Li Zhu2, Wei Zhao3, Daijie Chen2, Weiyi Huang1 and Sheng Yang3 1 2 3
College of Life Science, Nanjing Agriculture University, Nanjing, China School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Many strains of Amycolatopsis, such as Amycolatopsis orientalis, A. balhimycina, and A. mediterranei, are important antibiotic producers. Three indigenous plasmids, pMEA100, pMEA300, and pA387, found in this genus have been sequenced. However, only some vectors based on pA387 have been widely applied in Amycolatopsis research. An indigenous plasmid, pXL100, was isolated from the vancomycin producer A. orientalis HCCB10007. Sequence analysis of pXL100 revealed its total length to be 33,499 bp and GC content to be 68.9%. A 2830-bp fragment containing three ORFs has been identified as essential for replication in A. orientalis, but it has no significant similarity to any known replicons. A vector, pLYZW7-3, was constructed based on the pXL100 replicon and could be transferred into A. mediterranei and A. orientalis by electroporation or conjugation with high frequency. A mutant with a disrupted gene was successfully complemented with the pLYZW7-3 vector, indicating that the vector is potentially useful in Amycolatopsis research.
: Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Abbreviations: 2 YT – tryptone/yeast extract medium; bp – base pairs; CRM – complete precultivation medium; LB – Luria-Bertani medium; MS – soy/mannitol medium; ORF – open reading frame; rpm – revolutions per minute; TSB – tryptone soy broth Keywords: Amycolatopsis orientalis / Indigenous plasmid / Replicon / Shuttle vector
Received: March 10, 2014; accepted: July 12, 2014 DOI 10.1002/jobm.201400210
Introduction Amycolatopsis plays an important role in the antibiotics industry. Many strains in this genus produce antibiotics, such as vancomycin, balhimycin, and rifamycin. However, genetic manipulations of this genus are not easy because few vectors are useful, compared with those for Streptomyces. Until now, six indigenous plasmids have been found in the Amycolatopsis genus, but only three
Correspondence: Weiyi Huang, College of Life Science, Nanjing Agriculture University, NO.1 Weigang, Nanjing 210095, China; Sheng Yang, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, NO.300 Fenglin Road, Shanghai 200031, China E-mail: [email protected], [email protected] Phone/Fax: 862584395647, 862154924173 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
(pMEA100, pMEA300, and pA387) and their relevant vectors have been studied further. The pMEA100 plasmid (GenBank accession no. NC_010852, 23.3 kb) was isolated from Amycolatopsis mediterranei LBG A3136, a rifamycin producer, and it exists in an integrated as well as free form, whereas A. mediterranei ATCC 13685 only exists as an integrated form [1]. A cloning vector, pMEA123, containing 42% of the pMEA100 sequence was constructed, and resulted in one-third the transformation efficiency of pMEA100 [2]; however, no other study has described it. A. methanolica NCIB 11946 is a methanolutilizing bacterium that contains the pMEA300 plasmid (GenBank accession no. L36679, 13.3 kb), which is mostly maintained as an integrated element but can also replicate autonomously [3]. pMEA300-derived shuttle vectors capable of autonomous replication (korA, orfA,
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and orfB) have been constructed and have proven to be useful [4], particularly the pWV138 plasmid [5]. However, due to the host-range restriction, the vectors cannot be widely used. A. benzoatilytica (formerly A. orientalis) DSM 43387 contains a non-integrative plasmid, pA387 (GenBank accession no. EF375609, 30.2 kb) [6]. Various vectors based on the pA387 replicon have been constructed, and some have been applied in research on A. mediterranei and A. orientalis strains [7–10]; however, much work on the stability and operability of the vectors must be performed. It is necessary to develop new vector tools to be used in molecular research of the Amycolatopsis genus. An indigenous plasmid, pXL100, has been isolated from A. orientalis HCCB10007, which is a vancomycin producer. This plasmid has been sequenced using the Roche 454 GS FLX Titanium System at the Chinese National Human Genome Center at Shanghai. In this study, we characterized the pXL100 plasmid. A shuttle vector was first constructed based on the replicon of pXL100 and successfully applied in different Amycolatopsis strains with high efficiency.
Materials and methods Bacterial strains, plasmids, and growth media Table 1 shows the strains and plasmids used in this study. A. orientalis HCCB 10007 produces high yields of vancomycin and was derived from the species-type strain ATCC 43491 through a series of physical and chemical mutageneses. The pXL100 plasmid from this strain was isolated using a modified version of a previously mentioned protocol [7]. A. orientalis HCCB10007 and the plasmid-free strain A. orientalis ATCC43490 were grown at 28 °C in Gause’s synthetic agar medium [11], and the transformants and A. mediterranei were cultured in Bennet medium [12]. For vancomycin production, spores of A. orientalis and its derivative were inoculated into a 250-ml, baffled flask containing 25 ml of seed medium (20 g L1 glycerol, 40 g L1 amidulin, 20 g L1 soybean flour, 15 g L1 glucose, 6 g L1 KNO3, 0.2 g L1 KH2PO4, 0.4 g L1 MgCl2.6H2O), cultivated at 28 °C and 220 rpm for 44 h and then transferred to production medium (20 g L1 glycerol, 20 g L1 soybean flour, 6 g L1 KNO3, 0.2 g L1 KH2PO4, 0.4 g L1 MgCl2.6H2O, 3 g L1 CaCO3).
Table 1. Bacterial strains and plasmids used. Strain/Plasmid
Description
Source
A. orientalis HCCB10007 A. orientalis ATCC43490 A. mediterranei U32 E. coli JM110 E. coli ET12567(pUZ8002) pLYZL102 pXL100 pLYZW1
Vancomycin-producing strain, containing plasmid pXL100 Vancomycin-producing strain, lack of indigenous plasmid Rifamycin-producing strain dam dcm dam dcm hsdS 4.1 kb, oriT, aac(3)IV, bla, SceI site Indigenous plasmid from A. orientalis HCCB10007 pLYZL102 with insertion of the 12.9-kb KpnI/EcoRI fragment (positions 1342–14,312) of pXL100 pLYZL102 with insertion of the 18.5-kb EcoRV fragment (positions 7006–25,602) of pXL100 pLYZL102 with insertion of the 15.6-kb EcoRV fragment (positions 25,602–7006) of pXL100 pLYZL102 with insertion of the 12.5-kb ClaI fragment (positions 14,996–2,7518) of pXL100 pLYZL102 with insertion of the 7.9-kb ClaI fragment (positions 7002–14,996) of pXL100 pLYZL102 with insertion of the 5.5-kb ClaI fragment (positions 27,519–33,027) of pXL100 pLYZL102 with insertion of the 6.3-kb ClaI/KpnI fragment (positions 14,996–21,386) of pXL100 pLYZL102 with insertion of a 2947-bp fragment (positions 15,288–18,222) of pXL100 pLYZL102 with insertion of a 3012-bp fragment (positions 17,738–20,737) of pXL100 pLYZL102 with insertion of a 2830-bp fragment (positions 16,104–18,933) of pXL100 pLYZL102 with insertion of a 3542-bp fragment (bp 15,392–18,933) of pXL100 pLYZL102 with insertion of a 4308-bp fragment (positions 16,104–20,411) of pXL100 pLYZW7-3 with insertion of PermE and egfp pLYZW7-3 with insertion of PermE and gtfE
This lab ATCC [29] [30] [31] [23] This work This work
pLYZW2 pLYZW3 pLYZW4 pLYZW5 pLYZW6 pLYZW7 pLYZW7-1 pLYZW7-2 pLYZW7-3 pLYZW7-4 pLYZW7-5 pLYZWG pLYZWE
This work This work This work This work This work This work This This This This This This This
work work work work work work work
Note: ATCC, American type culture collection. PermE, promoter of the erythromycin resistance gene. egfp, the gene encoding green fluorescent protein. gtfE, a glycosyltransferase gene. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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DNA sequencing and annotation Plasmid sequencing was performed using the Roche 454 GS FLX Titanium System [13], and the putative protein coding sequences (CDSs) were predicted using Glimmer [14]. The gaps were filled using a PCR strategy. The final sequence assembly was performed using the phred/phrap/consed package (http://www.phrap.org/ phredphrapconsed.html). The CDSs annotation was based on the BLASTP results obtained using the KEGG, NR, and CDD databases.
10 mM MgCl2, 10 mM MgCl2) agar plates and grown at 28 °C for 16–20 h. Subsequently, the plates were overlaid with nalidixic acid (25 mg ml1) and apramycin (50 mg ml1) for selection.
Construction of the Escherichia coli-Amycolatopsis shuttle vector pLYZW7-3 Primer pairs ZW7-3F (50 -TCTAGATTACCTGCGGTTTCGCCGGTT-30 ) and ZW7-3R (50 -AAGCTTATGTTCTTCACCAGTGCAAC-30 ) (XbaI and HindIII sites, respectively, are represented in bold) were used to amplify a fragment of 2830 bp corresponding to the origin of pXL100 (pX-rep). The fragment was cloned into pUC19, excised, and linked using the appropriate enzymes and inserted into pLYZL102. The final plasmid was designated pLYZW7-3.
Results
Electroporation of plasmid DNA into Amycolatopsis strains The Amycolatopsis spores were inoculated into CRM liquid medium [15] and cultured at 28 °C. After 48 h, 2.5 ml of cell culture was added into 50 ml of fresh CRM medium plus 1.5% glycine and incubated for 16 h. Electrocompetent cells were collected and washed with cold water and buffer (0.3 M sucrose, 15% glycerol). Electroporation by a Gene Pulser Xcell (Bio-Rad, Inc.) was carried out using approximately 1–5 mg of DNA and 60 ml of competent cells (108 ufc ml1). Electroporation was performed in 2-mm, electrode-gap cuvettes at 7.5 kV cm1 for 15.3 ms. After the pulse, 1 ml of TSB medium (tryptone soy broth) was added directly, and the cells were revived at 28 °C for 6 h before being plated on selection medium. Conjugation of the vector from E. coli to A. orientalis E. coli ET12567 (pUZ8002) was chosen as the plasmid donor for conjugative transfer and was grown in LB medium [16] (25 mg ml1 kanamycin, 25 mg ml1 chloramphenicol, and 50 mg ml1 apramycin) at 37 °C (OD600 ¼ 0.5). The cells were harvested by centrifugation, washed twice, and resuspended in LB medium. A. orientalis was grown in Gause’s medium for 2 days, and the spores were selected on 10% glycerol. After the spores (107 ufc ml1) were resuspended in 2 YT medium, the donor cells were mixed with the recipients and incubated at 50 °C for 10 min. The mixture was spread on MS (20 g L1 mannose, 20 g L1 soybean flour, 20 g L1 agar, ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Nucleotide sequence accession number The complete nucleotide sequence of plasmid pXL100 from A. orientalis HCCB10007 has been deposited into the GenBank database under accession no. CP003411.
The sequence and analysis of pXL100 The indigenous plasmid pXL100 consists of 33,499 bp, and the GC content is 68.9%, which resembles that of the genome of A. orientalis (69%) [17]. The plasmids isolated from different species of Amycolatopsis are listed in Supporting Information Table S1. Sequence alignment of pXL100 with pA387, pMEA100, and pMEA300 revealed 48, 36.9, and 21.8% similarity, respectively, at the nucleotide level. The length of pXL100 is similar to that of pYO33 (33.5 kb, found in A. orientalis NRRL 2452/V33) [18]. Bioinformatics analysis revealed that the plasmid encodes 49 genes: 21 on the sense strand and 28 on the complementary strand (Fig. 1). The coding density is approximately 80.7%; however, only six ORFs resemble known genes (Table 2). Plasmid pXL100 is stably maintained in A. orientalis HCCB10007. According to the analysis of ORFs, two putative mechanisms of stable inheritance are present in this strain. AORIP_04c encodes a resolvase protein, which is a site-specific recombinase that resolves oligomers to monomers to achieve stable inheritance [19]. Another expressed protein is the partition A protein (ParA, AORIP_37), which has ATPase activity that provides energy for the process [20], but no ParB-like protein was identified. Interestingly, it was difficult to obtain a parA-disrupted mutant in our previous work (data not published), which suggests that pXL100-free A. orientalis HCCB10007 may not be viable. AORIP_28 encodes transcription factor WhiB, which is involved in a wide range of events, such as cell division, spore formation, nutrient starvation, and antibiotic resistance [21]. AORIP_35 has a conserved domain (COG1674) that is typically found within the DNA segregation ATPase FtsK/ SpoIIIE protein, which shows that the protein may be involved in the movement of DNA during conjugative transfer. Nudix hydrolases, whose function includes the housecleaning roles of eliminating toxic metabolites and controlling the availability of pathway intermediates, is encoded by AORIP_43c [22]. Additionally, the
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Figure 1. (A) The plasmid map of pXL100 from A. orientalis HCCB10007. The arrows indicate the positioning of the ORFs (AORIP_01AORIP_49) in circle 1. The positions of selected restriction sites are also indicated. Circle 2 depicts the GC content. Circle 3 shows the GC bias. (B) Electrophoretic mobility of digested pXL100 on an agarose gel. M: 1-kb marker; Lane 1: pXL100 digested by EcoRV; Lane 2: pXL100 digested by EcoRV and EcoRI.
pXL100 plasmid does not exhibit any significant DNA sequence homology to any characterized replication region, which indicates that this indigenous plasmid has a new replicon locus. Determination of the minimal replication region of pXL100 To investigate the replication of pXL100, various fragments were digested by endonucleases and cloned into pLYZL102 to obtain pLYZW1–pLYZW7. These clones represent the entire pXL100 plasmid. The plasmids were independently introduced into a plasmid-free strain, A. orientalis ATCC43490, by electroporation. Plasmids pLYZW2, pLYZW4, and pLYZW7 yielded transformants, whereas the others did not (Fig. 2). Plasmids were isolated from these transformants and separately transformed into E. coli JM110. The restriction patterns (Fig. 3) of the three plasmids isolated from E. coli appeared to be identical to the input plasmids, revealing that the replication region was located within the ClaI-to-KpnI fragment (6392 bp, positions 14996–21387) of pXL100. To delineate the essential region more closely, a set of derivate plasmids, pLYZW7-1–pLYZW7-5, of pLYZW7 were constructed. Figure 2 indicates that plasmids pLYZW7-3–pLYZW7-5 were able to replicate in ATCC43490, and all contained a fragment extending ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
from ORF AORIP_23 to AORIP_25. Thus the minimal replication region is 2830 bp in length (positions 16104– 18933). The three ORFs encoded proteins of 474, 70, and 280 amino acids, respectively. The nucleotide sequence of AORIP_23 from 556 to 1212 bp was similar (70% identity) to ORF7 and ORF8 of the pA387 plasmid. In addition, the 58-bp sequence (positions 1347–1404) shared 79% identity with ORF9 of pA387. The genes ORF8–ORF10 were sufficient for autonomous replication in pA387, whereas ORF7 was not necessary [6]. The nucleic acid sequence of AORIP_25 from 299 to 798 bp was 46% identical to ORF7 of pRL1 in Streptomyces sp. 44030. The product of AORIP_25 is a hypothetical protein. However, there was no similarity to AORIP_24 in any plasmids. Construction of the pLYZW7-3 shuttle vector To establish a conjunction system, an E. coli-Amycolatopsis shuttle vector pLYZW7-3 was constructed by cloning 2830 bp of the region necessary for replication into pLYZL102. Plasmid pLYZL102 [23] was constructed in our previous work and included the oriT fragment required for conjugal transfer from E. coli to Amycolatopsis. Another gene, aac(3)IV from pLYZL102, encoded resistance to apramycin and served as the selection marker. The plasmid pLYZW7-3 was approximately 7.4 kbp in length. In addition, six unique restriction sites (BglII/XbaI/HindIII/
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Table 2. Predicted ORFs in the complete sequence of pXL100.
AORIP_ORF
Position (bp)
Protein length
AORIP_01c AORIP_02c AORIP_03 AORIP_04c
137–769 988–1482 1780–2124 2260–2835
210 164 114 191
AORIP_05c AORIP_06 AORIP_07c AORIP_08 AORIP_09 AORIP_10 AORIP_11c AORIP_12c AORIP_13c
2835–3035 3587–4693 4684–5295 5455–5763 6251–6652 6907–7038 7235–8767 8764–9147 9979–10,500
66 368 203 102 133 43 510 127 173
AORIP_14c AORIP_15c AORIP_16c
11,021–11,446 11,452–12,213 12,210–12,728
141 253 172
AORIP_17c AORIP_18c AORIP_19c AORIP_20c AORIP_21c AORIP_22c AORIP_23c AORIP_24c AORIP_25c AORIP_26c AORIP_27 AORIP_28
12,728–13,702 13,699–14,379 14,376–14,846 14,937–15,386 15,392–15,715 15,742–15,945 16,104–17,528 17,816–18,028 18,091–18,933 18,944–19,786 19,989–20,411 20,465–20,704
324 226 156 149 107 67 474 70 280 280 140 79
AORIP_29 AORIP_30 AORIP_31 AORIP_32 AORIP_33 AORIP_34 AORIP_35
20,704–21,306 21,303–21,578 21,580–21,822 21,835–22,218 22,215–22,451 22,448–23,617 23,710–26,301
200 91 80 127 78 389 863
AORIP_36 AORIP_37 AORIP_38 AORIP_39c
26,573–27,229 27,445–28,248 28,369–28,653 28,725–28,991
218 267 94 88
AORIP_40 AORIP_41c AORIP_42c AORIP_43c
29,161–29,376 29,409–29,558 29,609–29,773 29,807–30,973
71 49 54 388
AORIP_44 AORIP_45 AORIP_46c AORIP_47 AORIP_48c AORIP_49c
31,195–31,383 31,470–31,745 31,738–31,998 32,218–32,526 32,542–32,967 33,020–33,472
62 91 86 102 141 150
Similarity or function (organism) Unknown Unknown Unknown Resolvase domain protein Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Hypothetical protein Unknown Unknown Predicted kinase Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Transcription factor WhiB Unknown Unknown Unknown Unknown Unknown Unknown Cell division FtsK/SpoIIIE Unknown parA Unknown Transcriptional regulator Unknown Unknown Unknown Nudix hydrolase Unknown Unknown Unknown Unknown Unknown Unknown
Figure 2. Identification of the replication region of pXL100. A linear map of the pXL100 restriction sites is shown at the top. The bars below the map represent each pXL100 fragment present in various plasmids. K: KpnI; E: EcoRI; Ev: EcoRV; C: ClaI.
ClaI/KpnI/EcoRI) can be used for cloning. A map of pLYZW7-3 is shown in Fig. 3. The pLYZW7-3 plasmid has a copy number of approximately 15 (Supporting Information Fig. S1) and does not integrate into the chromosome. Host range, transformation efficiency, and application To verify the replication and existence of pLYZW7-3 in the different strains, an enhanced green fluorescent protein (EGFP) reporter gene was cloned into pLYZW7-3 to yield pLYZWG. The egfp gene is a modified version of the green fluorescent protein (GFP) gene of the jellyfish
Figure 3. The restriction pattern of the three isolated plasmids (pLYZW2, pLYZW4, and pLYZW7). M: 1-kb marker; Lane1: pLYZW2 digested by EcoRI and EcoRV; Lane2: pLYZW4 digested by EcoRI and EcoRV; Lane3: pLYZW7 digested by KpnI and EcoRV. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Aequorea victoria that has a codon usage that corresponds well to that found in many GC-rich Streptomycete genes [24]. The expression of the egfp gene was induced by the erythromycin resistance (PermE) promoter when pLYZWG replicated in the strains. All of the correct clones were identified by monitoring of fluorescence in hyphae on a microscope slide (Fig. 4), which suggested the replication of pLYZW7-3 in A. orientalis and A. mediterranei. On the other hand, the use of EGFP as a reporter for gene expression in A. orientalis
was demonstrated. The pLYZWG plasmid and pA387derived pULVK2A vector was transferred into A. orientalis ATCC43490, A. mediterranei U32, and A. orientalis HCCB10007 by electroporation. We compared their transformation efficiency, and found that both were highly efficient (Table 3). To establish the conjugation efficiency of pLYZW7-3, E. coli ET12567 (pUZ8002) was used as the donor strain. A. orientalis ATCC43490, A. orientalis HCCB10007, Streptomyces coelicolor M145, and Streptomyces lividans TK23 were
Figure 4. The map of the shuttle vector pLYZW7-3 and its applications. EGFP as a reporter was used to verify the replication of the vector in different host-ranges. Under the fluorescence microscopy the strains containing pLYZWG appeared green, and the control group (strains without pLYZWG) did not fluoresce (top). The HPLC spectra of the pLYZW7-3 complemented mutant (A. orientalis DE) with a disrupted gtfE gene (bottom). oriT, origin of transfer of plasmid pIJ773; bla, ampicillin resistance gene; acc(3)IV, apramycin resistance gene; pX-rep, replication origin of pXL100; PermE, promoter of the erythromycin resistance gene; egfp, enhanced green fluorescent protein gene; gtfE, glycosyltransferase gene; scale bar, 50 mm.
Table 3. Host range and transformation frequency of the vectors. Transformation frequencya pXL100-derived vector pLYZW7-3
pA387-derived vector pULVK2A
Species and strain
Conjugation (/cfu)
Electroporation (transf/mg)
Conjugation
Electroporation (transf/mg)
A. orientalis ATCC43490 A. orientalis HCCB10007 A. mediterranei U32 S. coelicolor M145 S. lividans TK23
1 105 – 0 0
1.8 105 1.6 105 1.5 105 – –
– – – – –
1.2 105 1 105 2 105 – –
Conjugation is measured in terms of conjugation frequency per recipient, and transformants per mg (transf/mg) for electroporation.
a
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used as the recipients for conjugation. The vector was first introduced into E. coli ET12567 (pUZ8002) and then transferred into the recipients, which resulted in 100 apramycin-resistant clones of A. orientalis on each plate (Table 3). However, no Streptomyces clone was obtained. The transformation methods for pLYZW7-3 were confirmed to be useful, which indicated that the vector could be used in certain hosts. To further investigate the applicability of the pLYZW73, vector the pLYZWE plasmid was constructed by cloning a glycosyltransferase gene, gtfE, from the vancomycin gene cluster into the BglII site of pLYZW73. Then, the pLYZWE plasmid was introduced into A. orientalis DE (DgtfE, only producing aglucovancomycin). The obtained recombinant (A. orientalis/pLYZWE) recovered the production of vancomycin (Fig. 4), confirming pLYZW7-3 as a potentially useful molecular vector in Amycolatopsis research.
Discussion Strains of A. orientalis are the producers of the important antibiotic vancomycin. However, to our knowledge, it is not easy to genetically manipulate these strains. An indigenous plasmid isolated from A. orientalis HCCB10007, pXL100, has been sequenced and analyzed, and new vector tools were constructed to overcome the difficulties of transformation. Nucleotide sequence analysis revealed that plasmid pXL100 shared low homology with three known plasmids (pMEA100, pMEA300, pA387) in Amycolatopsis, and most genes in pXL100 were completely unknown. The sequence of pXL100 provides a crucial reference for comparative analysis with other actinomycetes plasmids. Compared to the cleavage maps of pYO33 isolated from A. orientalis NRRL 2452/V33, the positions of restricted sites for the enzymes EcoRI/EcoRV were the same, but for other restricted enzymes, such as BamHI, XhoI, and NaeI, were apparently different. However, sequences related to integrative or free plasmids were not found in A. orientalis subsp. orientalis strain KCTC 9412, which is another vancomycin producer whose genome was recently sequenced [25]. These findings suggest that pXL100 is novel and the first sequenced plasmid of A. orientalis strains. Plasmids are usually comprised of genes required for replication, maintenance, and transfer. Bioinformatic analysis of pXL100 showed no homology to known replication regions, suggesting the replicon was unique. Two putative proteins (resolvase and ParA), which are related to stable inheritance, were identified, and functional analysis revealed a gene that was responsible ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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for conjugal transfer, the function of which was similar to the chromosomal DNA translocators FtsK and SpoIIIE. It can be speculated that the strain uses a segregation-like transfer mechanism, similar to pSVH1 in Streptomyces [26] and pCW3 in Clostridium perfringens [27]. The pMEA-like plasmids are maintained as an integrated form and as a free form when pA387 and pXL100 replicate autonomously. Te Poele et al. [28] indicated that the replication element of the pMEAsequence (repAM) evolved in an integrated form rather than by horizontal gene transfer of the free-replicating form. We hypothesize that the replication regions of pA387 and pXL100 might have evolved by horizontal gene transfer. On the other hand, the replication region (AORIP_23AORIP_25) of pXL100 was identified, and analysis revealed that only AORIP_23 was partially similar to the known pA387 replicon. An E. coli-Amycolatopsis shuttle vector, pLYZW7-3, was first constructed based on this replicon. This vector was capable of being transferred to certain Amycolatopsis hosts by electroporation or conjugation, and it replicated in these strains. The transformation frequency of pLYZW7-3 was nearly the same as that of the widely used plasmid, pULVK2A. The pLYZW73 plasmid, as an expression plasmid, also successfully expressed the glycosyltransferase gene in Amycolatopsis strains. Based upon our results, plasmid pLYZW7-3 could become a useful tool for genetic manipulation in Amycolatopsis research.
Acknowledgments This work was supported by grants from the National High Technology Research and Development Program of China (2012AA02706).
Conflict of interest statement All authors have no financial/commercial conflicts of interest.
References [1] Moretti, P., Hintermann, G., Hütter, R., 1985. Isolation and characterization of an extra chromosomal element from Nocardia mediterranei. Plasmid, 14, 126–133. [2] Madoń, J., Hütter, R., 1991. Transformation system for Amycolatopsis (Nocardia) mediterranei: direct transformation of mycelium with plasmid DNA. J. Bacteriol., 173, 6325– 6331.
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Research Paper Characterization of plasmid pXL100 from Amycolatopsis orientalis HCCB10007 and construction of a shuttle vector Li Xu1,2, Yanmei Li2, Li Zhu2, Wei Zhao3, Daijie Chen2, Weiyi Huang1 and Sheng Yang3 1 2 3
College of Life Science, Nanjing Agriculture University, Nanjing, China School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Many strains of Amycolatopsis, such as Amycolatopsis orientalis, A. balhimycina, and A. mediterranei, are important antibiotic producers. Three indigenous plasmids, pMEA100, pMEA300, and pA387, found in this genus have been sequenced. However, only some vectors based on pA387 have been widely applied in Amycolatopsis research. An indigenous plasmid, pXL100, was isolated from the vancomycin producer A. orientalis HCCB10007. Sequence analysis of pXL100 revealed its total length to be 33,499 bp and GC content to be 68.9%. A 2830-bp fragment containing three ORFs has been identified as essential for replication in A. orientalis, but it has no significant similarity to any known replicons. A vector, pLYZW7-3, was constructed based on the pXL100 replicon and could be transferred into A. mediterranei and A. orientalis by electroporation or conjugation with high frequency. A mutant with a disrupted gene was successfully complemented with the pLYZW7-3 vector, indicating that the vector is potentially useful in Amycolatopsis research.
: Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Abbreviations: 2 YT – tryptone/yeast extract medium; bp – base pairs; CRM – complete precultivation medium; LB – Luria-Bertani medium; MS – soy/mannitol medium; ORF – open reading frame; rpm – revolutions per minute; TSB – tryptone soy broth Keywords: Amycolatopsis orientalis / Indigenous plasmid / Replicon / Shuttle vector
Received: March 10, 2014; accepted: July 12, 2014 DOI 10.1002/jobm.201400210
Introduction Amycolatopsis plays an important role in the antibiotics industry. Many strains in this genus produce antibiotics, such as vancomycin, balhimycin, and rifamycin. However, genetic manipulations of this genus are not easy because few vectors are useful, compared with those for Streptomyces. Until now, six indigenous plasmids have been found in the Amycolatopsis genus, but only three
Correspondence: Weiyi Huang, College of Life Science, Nanjing Agriculture University, NO.1 Weigang, Nanjing 210095, China; Sheng Yang, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, NO.300 Fenglin Road, Shanghai 200031, China E-mail: [email protected], [email protected] Phone/Fax: 862584395647, 862154924173 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
(pMEA100, pMEA300, and pA387) and their relevant vectors have been studied further. The pMEA100 plasmid (GenBank accession no. NC_010852, 23.3 kb) was isolated from Amycolatopsis mediterranei LBG A3136, a rifamycin producer, and it exists in an integrated as well as free form, whereas A. mediterranei ATCC 13685 only exists as an integrated form [1]. A cloning vector, pMEA123, containing 42% of the pMEA100 sequence was constructed, and resulted in one-third the transformation efficiency of pMEA100 [2]; however, no other study has described it. A. methanolica NCIB 11946 is a methanolutilizing bacterium that contains the pMEA300 plasmid (GenBank accession no. L36679, 13.3 kb), which is mostly maintained as an integrated element but can also replicate autonomously [3]. pMEA300-derived shuttle vectors capable of autonomous replication (korA, orfA,
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and orfB) have been constructed and have proven to be useful [4], particularly the pWV138 plasmid [5]. However, due to the host-range restriction, the vectors cannot be widely used. A. benzoatilytica (formerly A. orientalis) DSM 43387 contains a non-integrative plasmid, pA387 (GenBank accession no. EF375609, 30.2 kb) [6]. Various vectors based on the pA387 replicon have been constructed, and some have been applied in research on A. mediterranei and A. orientalis strains [7–10]; however, much work on the stability and operability of the vectors must be performed. It is necessary to develop new vector tools to be used in molecular research of the Amycolatopsis genus. An indigenous plasmid, pXL100, has been isolated from A. orientalis HCCB10007, which is a vancomycin producer. This plasmid has been sequenced using the Roche 454 GS FLX Titanium System at the Chinese National Human Genome Center at Shanghai. In this study, we characterized the pXL100 plasmid. A shuttle vector was first constructed based on the replicon of pXL100 and successfully applied in different Amycolatopsis strains with high efficiency.
Materials and methods Bacterial strains, plasmids, and growth media Table 1 shows the strains and plasmids used in this study. A. orientalis HCCB 10007 produces high yields of vancomycin and was derived from the species-type strain ATCC 43491 through a series of physical and chemical mutageneses. The pXL100 plasmid from this strain was isolated using a modified version of a previously mentioned protocol [7]. A. orientalis HCCB10007 and the plasmid-free strain A. orientalis ATCC43490 were grown at 28 °C in Gause’s synthetic agar medium [11], and the transformants and A. mediterranei were cultured in Bennet medium [12]. For vancomycin production, spores of A. orientalis and its derivative were inoculated into a 250-ml, baffled flask containing 25 ml of seed medium (20 g L1 glycerol, 40 g L1 amidulin, 20 g L1 soybean flour, 15 g L1 glucose, 6 g L1 KNO3, 0.2 g L1 KH2PO4, 0.4 g L1 MgCl2.6H2O), cultivated at 28 °C and 220 rpm for 44 h and then transferred to production medium (20 g L1 glycerol, 20 g L1 soybean flour, 6 g L1 KNO3, 0.2 g L1 KH2PO4, 0.4 g L1 MgCl2.6H2O, 3 g L1 CaCO3).
Table 1. Bacterial strains and plasmids used. Strain/Plasmid
Description
Source
A. orientalis HCCB10007 A. orientalis ATCC43490 A. mediterranei U32 E. coli JM110 E. coli ET12567(pUZ8002) pLYZL102 pXL100 pLYZW1
Vancomycin-producing strain, containing plasmid pXL100 Vancomycin-producing strain, lack of indigenous plasmid Rifamycin-producing strain dam dcm dam dcm hsdS 4.1 kb, oriT, aac(3)IV, bla, SceI site Indigenous plasmid from A. orientalis HCCB10007 pLYZL102 with insertion of the 12.9-kb KpnI/EcoRI fragment (positions 1342–14,312) of pXL100 pLYZL102 with insertion of the 18.5-kb EcoRV fragment (positions 7006–25,602) of pXL100 pLYZL102 with insertion of the 15.6-kb EcoRV fragment (positions 25,602–7006) of pXL100 pLYZL102 with insertion of the 12.5-kb ClaI fragment (positions 14,996–2,7518) of pXL100 pLYZL102 with insertion of the 7.9-kb ClaI fragment (positions 7002–14,996) of pXL100 pLYZL102 with insertion of the 5.5-kb ClaI fragment (positions 27,519–33,027) of pXL100 pLYZL102 with insertion of the 6.3-kb ClaI/KpnI fragment (positions 14,996–21,386) of pXL100 pLYZL102 with insertion of a 2947-bp fragment (positions 15,288–18,222) of pXL100 pLYZL102 with insertion of a 3012-bp fragment (positions 17,738–20,737) of pXL100 pLYZL102 with insertion of a 2830-bp fragment (positions 16,104–18,933) of pXL100 pLYZL102 with insertion of a 3542-bp fragment (bp 15,392–18,933) of pXL100 pLYZL102 with insertion of a 4308-bp fragment (positions 16,104–20,411) of pXL100 pLYZW7-3 with insertion of PermE and egfp pLYZW7-3 with insertion of PermE and gtfE
This lab ATCC [29] [30] [31] [23] This work This work
pLYZW2 pLYZW3 pLYZW4 pLYZW5 pLYZW6 pLYZW7 pLYZW7-1 pLYZW7-2 pLYZW7-3 pLYZW7-4 pLYZW7-5 pLYZWG pLYZWE
This work This work This work This work This work This work This This This This This This This
work work work work work work work
Note: ATCC, American type culture collection. PermE, promoter of the erythromycin resistance gene. egfp, the gene encoding green fluorescent protein. gtfE, a glycosyltransferase gene. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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DNA sequencing and annotation Plasmid sequencing was performed using the Roche 454 GS FLX Titanium System [13], and the putative protein coding sequences (CDSs) were predicted using Glimmer [14]. The gaps were filled using a PCR strategy. The final sequence assembly was performed using the phred/phrap/consed package (http://www.phrap.org/ phredphrapconsed.html). The CDSs annotation was based on the BLASTP results obtained using the KEGG, NR, and CDD databases.
10 mM MgCl2, 10 mM MgCl2) agar plates and grown at 28 °C for 16–20 h. Subsequently, the plates were overlaid with nalidixic acid (25 mg ml1) and apramycin (50 mg ml1) for selection.
Construction of the Escherichia coli-Amycolatopsis shuttle vector pLYZW7-3 Primer pairs ZW7-3F (50 -TCTAGATTACCTGCGGTTTCGCCGGTT-30 ) and ZW7-3R (50 -AAGCTTATGTTCTTCACCAGTGCAAC-30 ) (XbaI and HindIII sites, respectively, are represented in bold) were used to amplify a fragment of 2830 bp corresponding to the origin of pXL100 (pX-rep). The fragment was cloned into pUC19, excised, and linked using the appropriate enzymes and inserted into pLYZL102. The final plasmid was designated pLYZW7-3.
Results
Electroporation of plasmid DNA into Amycolatopsis strains The Amycolatopsis spores were inoculated into CRM liquid medium [15] and cultured at 28 °C. After 48 h, 2.5 ml of cell culture was added into 50 ml of fresh CRM medium plus 1.5% glycine and incubated for 16 h. Electrocompetent cells were collected and washed with cold water and buffer (0.3 M sucrose, 15% glycerol). Electroporation by a Gene Pulser Xcell (Bio-Rad, Inc.) was carried out using approximately 1–5 mg of DNA and 60 ml of competent cells (108 ufc ml1). Electroporation was performed in 2-mm, electrode-gap cuvettes at 7.5 kV cm1 for 15.3 ms. After the pulse, 1 ml of TSB medium (tryptone soy broth) was added directly, and the cells were revived at 28 °C for 6 h before being plated on selection medium. Conjugation of the vector from E. coli to A. orientalis E. coli ET12567 (pUZ8002) was chosen as the plasmid donor for conjugative transfer and was grown in LB medium [16] (25 mg ml1 kanamycin, 25 mg ml1 chloramphenicol, and 50 mg ml1 apramycin) at 37 °C (OD600 ¼ 0.5). The cells were harvested by centrifugation, washed twice, and resuspended in LB medium. A. orientalis was grown in Gause’s medium for 2 days, and the spores were selected on 10% glycerol. After the spores (107 ufc ml1) were resuspended in 2 YT medium, the donor cells were mixed with the recipients and incubated at 50 °C for 10 min. The mixture was spread on MS (20 g L1 mannose, 20 g L1 soybean flour, 20 g L1 agar, ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Nucleotide sequence accession number The complete nucleotide sequence of plasmid pXL100 from A. orientalis HCCB10007 has been deposited into the GenBank database under accession no. CP003411.
The sequence and analysis of pXL100 The indigenous plasmid pXL100 consists of 33,499 bp, and the GC content is 68.9%, which resembles that of the genome of A. orientalis (69%) [17]. The plasmids isolated from different species of Amycolatopsis are listed in Supporting Information Table S1. Sequence alignment of pXL100 with pA387, pMEA100, and pMEA300 revealed 48, 36.9, and 21.8% similarity, respectively, at the nucleotide level. The length of pXL100 is similar to that of pYO33 (33.5 kb, found in A. orientalis NRRL 2452/V33) [18]. Bioinformatics analysis revealed that the plasmid encodes 49 genes: 21 on the sense strand and 28 on the complementary strand (Fig. 1). The coding density is approximately 80.7%; however, only six ORFs resemble known genes (Table 2). Plasmid pXL100 is stably maintained in A. orientalis HCCB10007. According to the analysis of ORFs, two putative mechanisms of stable inheritance are present in this strain. AORIP_04c encodes a resolvase protein, which is a site-specific recombinase that resolves oligomers to monomers to achieve stable inheritance [19]. Another expressed protein is the partition A protein (ParA, AORIP_37), which has ATPase activity that provides energy for the process [20], but no ParB-like protein was identified. Interestingly, it was difficult to obtain a parA-disrupted mutant in our previous work (data not published), which suggests that pXL100-free A. orientalis HCCB10007 may not be viable. AORIP_28 encodes transcription factor WhiB, which is involved in a wide range of events, such as cell division, spore formation, nutrient starvation, and antibiotic resistance [21]. AORIP_35 has a conserved domain (COG1674) that is typically found within the DNA segregation ATPase FtsK/ SpoIIIE protein, which shows that the protein may be involved in the movement of DNA during conjugative transfer. Nudix hydrolases, whose function includes the housecleaning roles of eliminating toxic metabolites and controlling the availability of pathway intermediates, is encoded by AORIP_43c [22]. Additionally, the
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Figure 1. (A) The plasmid map of pXL100 from A. orientalis HCCB10007. The arrows indicate the positioning of the ORFs (AORIP_01AORIP_49) in circle 1. The positions of selected restriction sites are also indicated. Circle 2 depicts the GC content. Circle 3 shows the GC bias. (B) Electrophoretic mobility of digested pXL100 on an agarose gel. M: 1-kb marker; Lane 1: pXL100 digested by EcoRV; Lane 2: pXL100 digested by EcoRV and EcoRI.
pXL100 plasmid does not exhibit any significant DNA sequence homology to any characterized replication region, which indicates that this indigenous plasmid has a new replicon locus. Determination of the minimal replication region of pXL100 To investigate the replication of pXL100, various fragments were digested by endonucleases and cloned into pLYZL102 to obtain pLYZW1–pLYZW7. These clones represent the entire pXL100 plasmid. The plasmids were independently introduced into a plasmid-free strain, A. orientalis ATCC43490, by electroporation. Plasmids pLYZW2, pLYZW4, and pLYZW7 yielded transformants, whereas the others did not (Fig. 2). Plasmids were isolated from these transformants and separately transformed into E. coli JM110. The restriction patterns (Fig. 3) of the three plasmids isolated from E. coli appeared to be identical to the input plasmids, revealing that the replication region was located within the ClaI-to-KpnI fragment (6392 bp, positions 14996–21387) of pXL100. To delineate the essential region more closely, a set of derivate plasmids, pLYZW7-1–pLYZW7-5, of pLYZW7 were constructed. Figure 2 indicates that plasmids pLYZW7-3–pLYZW7-5 were able to replicate in ATCC43490, and all contained a fragment extending ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
from ORF AORIP_23 to AORIP_25. Thus the minimal replication region is 2830 bp in length (positions 16104– 18933). The three ORFs encoded proteins of 474, 70, and 280 amino acids, respectively. The nucleotide sequence of AORIP_23 from 556 to 1212 bp was similar (70% identity) to ORF7 and ORF8 of the pA387 plasmid. In addition, the 58-bp sequence (positions 1347–1404) shared 79% identity with ORF9 of pA387. The genes ORF8–ORF10 were sufficient for autonomous replication in pA387, whereas ORF7 was not necessary [6]. The nucleic acid sequence of AORIP_25 from 299 to 798 bp was 46% identical to ORF7 of pRL1 in Streptomyces sp. 44030. The product of AORIP_25 is a hypothetical protein. However, there was no similarity to AORIP_24 in any plasmids. Construction of the pLYZW7-3 shuttle vector To establish a conjunction system, an E. coli-Amycolatopsis shuttle vector pLYZW7-3 was constructed by cloning 2830 bp of the region necessary for replication into pLYZL102. Plasmid pLYZL102 [23] was constructed in our previous work and included the oriT fragment required for conjugal transfer from E. coli to Amycolatopsis. Another gene, aac(3)IV from pLYZL102, encoded resistance to apramycin and served as the selection marker. The plasmid pLYZW7-3 was approximately 7.4 kbp in length. In addition, six unique restriction sites (BglII/XbaI/HindIII/
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Table 2. Predicted ORFs in the complete sequence of pXL100.
AORIP_ORF
Position (bp)
Protein length
AORIP_01c AORIP_02c AORIP_03 AORIP_04c
137–769 988–1482 1780–2124 2260–2835
210 164 114 191
AORIP_05c AORIP_06 AORIP_07c AORIP_08 AORIP_09 AORIP_10 AORIP_11c AORIP_12c AORIP_13c
2835–3035 3587–4693 4684–5295 5455–5763 6251–6652 6907–7038 7235–8767 8764–9147 9979–10,500
66 368 203 102 133 43 510 127 173
AORIP_14c AORIP_15c AORIP_16c
11,021–11,446 11,452–12,213 12,210–12,728
141 253 172
AORIP_17c AORIP_18c AORIP_19c AORIP_20c AORIP_21c AORIP_22c AORIP_23c AORIP_24c AORIP_25c AORIP_26c AORIP_27 AORIP_28
12,728–13,702 13,699–14,379 14,376–14,846 14,937–15,386 15,392–15,715 15,742–15,945 16,104–17,528 17,816–18,028 18,091–18,933 18,944–19,786 19,989–20,411 20,465–20,704
324 226 156 149 107 67 474 70 280 280 140 79
AORIP_29 AORIP_30 AORIP_31 AORIP_32 AORIP_33 AORIP_34 AORIP_35
20,704–21,306 21,303–21,578 21,580–21,822 21,835–22,218 22,215–22,451 22,448–23,617 23,710–26,301
200 91 80 127 78 389 863
AORIP_36 AORIP_37 AORIP_38 AORIP_39c
26,573–27,229 27,445–28,248 28,369–28,653 28,725–28,991
218 267 94 88
AORIP_40 AORIP_41c AORIP_42c AORIP_43c
29,161–29,376 29,409–29,558 29,609–29,773 29,807–30,973
71 49 54 388
AORIP_44 AORIP_45 AORIP_46c AORIP_47 AORIP_48c AORIP_49c
31,195–31,383 31,470–31,745 31,738–31,998 32,218–32,526 32,542–32,967 33,020–33,472
62 91 86 102 141 150
Similarity or function (organism) Unknown Unknown Unknown Resolvase domain protein Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Hypothetical protein Unknown Unknown Predicted kinase Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Transcription factor WhiB Unknown Unknown Unknown Unknown Unknown Unknown Cell division FtsK/SpoIIIE Unknown parA Unknown Transcriptional regulator Unknown Unknown Unknown Nudix hydrolase Unknown Unknown Unknown Unknown Unknown Unknown
Figure 2. Identification of the replication region of pXL100. A linear map of the pXL100 restriction sites is shown at the top. The bars below the map represent each pXL100 fragment present in various plasmids. K: KpnI; E: EcoRI; Ev: EcoRV; C: ClaI.
ClaI/KpnI/EcoRI) can be used for cloning. A map of pLYZW7-3 is shown in Fig. 3. The pLYZW7-3 plasmid has a copy number of approximately 15 (Supporting Information Fig. S1) and does not integrate into the chromosome. Host range, transformation efficiency, and application To verify the replication and existence of pLYZW7-3 in the different strains, an enhanced green fluorescent protein (EGFP) reporter gene was cloned into pLYZW7-3 to yield pLYZWG. The egfp gene is a modified version of the green fluorescent protein (GFP) gene of the jellyfish
Figure 3. The restriction pattern of the three isolated plasmids (pLYZW2, pLYZW4, and pLYZW7). M: 1-kb marker; Lane1: pLYZW2 digested by EcoRI and EcoRV; Lane2: pLYZW4 digested by EcoRI and EcoRV; Lane3: pLYZW7 digested by KpnI and EcoRV. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Aequorea victoria that has a codon usage that corresponds well to that found in many GC-rich Streptomycete genes [24]. The expression of the egfp gene was induced by the erythromycin resistance (PermE) promoter when pLYZWG replicated in the strains. All of the correct clones were identified by monitoring of fluorescence in hyphae on a microscope slide (Fig. 4), which suggested the replication of pLYZW7-3 in A. orientalis and A. mediterranei. On the other hand, the use of EGFP as a reporter for gene expression in A. orientalis
was demonstrated. The pLYZWG plasmid and pA387derived pULVK2A vector was transferred into A. orientalis ATCC43490, A. mediterranei U32, and A. orientalis HCCB10007 by electroporation. We compared their transformation efficiency, and found that both were highly efficient (Table 3). To establish the conjugation efficiency of pLYZW7-3, E. coli ET12567 (pUZ8002) was used as the donor strain. A. orientalis ATCC43490, A. orientalis HCCB10007, Streptomyces coelicolor M145, and Streptomyces lividans TK23 were
Figure 4. The map of the shuttle vector pLYZW7-3 and its applications. EGFP as a reporter was used to verify the replication of the vector in different host-ranges. Under the fluorescence microscopy the strains containing pLYZWG appeared green, and the control group (strains without pLYZWG) did not fluoresce (top). The HPLC spectra of the pLYZW7-3 complemented mutant (A. orientalis DE) with a disrupted gtfE gene (bottom). oriT, origin of transfer of plasmid pIJ773; bla, ampicillin resistance gene; acc(3)IV, apramycin resistance gene; pX-rep, replication origin of pXL100; PermE, promoter of the erythromycin resistance gene; egfp, enhanced green fluorescent protein gene; gtfE, glycosyltransferase gene; scale bar, 50 mm.
Table 3. Host range and transformation frequency of the vectors. Transformation frequencya pXL100-derived vector pLYZW7-3
pA387-derived vector pULVK2A
Species and strain
Conjugation (/cfu)
Electroporation (transf/mg)
Conjugation
Electroporation (transf/mg)
A. orientalis ATCC43490 A. orientalis HCCB10007 A. mediterranei U32 S. coelicolor M145 S. lividans TK23
1 105 – 0 0
1.8 105 1.6 105 1.5 105 – –
– – – – –
1.2 105 1 105 2 105 – –
Conjugation is measured in terms of conjugation frequency per recipient, and transformants per mg (transf/mg) for electroporation.
a
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J. Basic Microbiol. 2015, 55, 247–254
Characterization of plasmid pXL100 and construction of a shuttle vector
used as the recipients for conjugation. The vector was first introduced into E. coli ET12567 (pUZ8002) and then transferred into the recipients, which resulted in 100 apramycin-resistant clones of A. orientalis on each plate (Table 3). However, no Streptomyces clone was obtained. The transformation methods for pLYZW7-3 were confirmed to be useful, which indicated that the vector could be used in certain hosts. To further investigate the applicability of the pLYZW73, vector the pLYZWE plasmid was constructed by cloning a glycosyltransferase gene, gtfE, from the vancomycin gene cluster into the BglII site of pLYZW73. Then, the pLYZWE plasmid was introduced into A. orientalis DE (DgtfE, only producing aglucovancomycin). The obtained recombinant (A. orientalis/pLYZWE) recovered the production of vancomycin (Fig. 4), confirming pLYZW7-3 as a potentially useful molecular vector in Amycolatopsis research.
Discussion Strains of A. orientalis are the producers of the important antibiotic vancomycin. However, to our knowledge, it is not easy to genetically manipulate these strains. An indigenous plasmid isolated from A. orientalis HCCB10007, pXL100, has been sequenced and analyzed, and new vector tools were constructed to overcome the difficulties of transformation. Nucleotide sequence analysis revealed that plasmid pXL100 shared low homology with three known plasmids (pMEA100, pMEA300, pA387) in Amycolatopsis, and most genes in pXL100 were completely unknown. The sequence of pXL100 provides a crucial reference for comparative analysis with other actinomycetes plasmids. Compared to the cleavage maps of pYO33 isolated from A. orientalis NRRL 2452/V33, the positions of restricted sites for the enzymes EcoRI/EcoRV were the same, but for other restricted enzymes, such as BamHI, XhoI, and NaeI, were apparently different. However, sequences related to integrative or free plasmids were not found in A. orientalis subsp. orientalis strain KCTC 9412, which is another vancomycin producer whose genome was recently sequenced [25]. These findings suggest that pXL100 is novel and the first sequenced plasmid of A. orientalis strains. Plasmids are usually comprised of genes required for replication, maintenance, and transfer. Bioinformatic analysis of pXL100 showed no homology to known replication regions, suggesting the replicon was unique. Two putative proteins (resolvase and ParA), which are related to stable inheritance, were identified, and functional analysis revealed a gene that was responsible ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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for conjugal transfer, the function of which was similar to the chromosomal DNA translocators FtsK and SpoIIIE. It can be speculated that the strain uses a segregation-like transfer mechanism, similar to pSVH1 in Streptomyces [26] and pCW3 in Clostridium perfringens [27]. The pMEA-like plasmids are maintained as an integrated form and as a free form when pA387 and pXL100 replicate autonomously. Te Poele et al. [28] indicated that the replication element of the pMEAsequence (repAM) evolved in an integrated form rather than by horizontal gene transfer of the free-replicating form. We hypothesize that the replication regions of pA387 and pXL100 might have evolved by horizontal gene transfer. On the other hand, the replication region (AORIP_23AORIP_25) of pXL100 was identified, and analysis revealed that only AORIP_23 was partially similar to the known pA387 replicon. An E. coli-Amycolatopsis shuttle vector, pLYZW7-3, was first constructed based on this replicon. This vector was capable of being transferred to certain Amycolatopsis hosts by electroporation or conjugation, and it replicated in these strains. The transformation frequency of pLYZW7-3 was nearly the same as that of the widely used plasmid, pULVK2A. The pLYZW73 plasmid, as an expression plasmid, also successfully expressed the glycosyltransferase gene in Amycolatopsis strains. Based upon our results, plasmid pLYZW7-3 could become a useful tool for genetic manipulation in Amycolatopsis research.
Acknowledgments This work was supported by grants from the National High Technology Research and Development Program of China (2012AA02706).
Conflict of interest statement All authors have no financial/commercial conflicts of interest.
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