Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Correspondence: Yuan Li, Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China. Tel.: +86 10 63153320;
Ebosin is a novel exopolysaccharide produced by Streptomyces sp. 139 with remarkable antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste) consisting of 27 ORFs has been identified. For functional analysis, one of the ste genes, ste9, was disrupted and then the gene complementation was performed. The resultant mutant Streptomyces sp. 139 (ste9−) produced polysaccharides with molecular weights of about 4.153 × 105 which is much smaller than that of Ebosin (9.03 × 105). The complemented strain Streptomyces sp. 139 (pKC9c) showed recovery in the molecular weights of EPS produced (8.004 × 105). As the theoretical protein product of ste9 is a chain length determinant (Wzz) homologue by sequence similarity, ste9 was cloned and expressed in E. coli 086:H2 (wzz−) for a complementation test. SDS-PAGE analysis showed that E. coli 086:H2 (wzz−) (pET30a-ste9) produced a modal chain length lipid polysaccharide (LPS) similar to that of the wild-type E. coli 086:H2. In addition, the expression of ste9 was able to restore the serum resistance of E. coli 086:H2 (wzz−) to almost the level of the wild-type strain. These results indicate that the ste9 gene is coding for a chain length determinant which plays an important role in Ebosin biosynthesis.
Microbial polysaccharides can be present as constituents of cell walls, as parts of lipopolysaccharides (LPSs) often referred to as O antigens, as capsular polysaccharides (CPSs) associated with the cell surface, or they can be secreted as exopolysaccharide (EPS). Bacterial biosynthesis of EPSs starts with the intracellular formation of a repeating unit on a lipid carrier, which is located in the cytoplasmic membrane. The later steps involve transport of the repeating units across the membrane to the outer layer and polymerization of several hundred to several thousand such units to form the final EPSs (Sikkema & Oba, 1998).
Bacterial LPS typically consists of three structural parts: lipid A, core oligosaccharide, and O antigen. Before ligation to lipid A-core, O antigen is synthesized separately to form LPS. The O unit is synthesized by sequential transfer of sugars from respective sugar nucleotides to the carrier lipid, undecaprenol phosphate (UndP) at the cytoplasmic side of the inner membrane. The number of O units attached to the lipid A-core is regulated by the chain length determinant Wzz (Franco et al., 1998). Wzz proteins belong to the ‘polysaccharide co-polymerases’ (PCP) super family, member of which are involved in the chain length regulation of polysaccharides including O antigen, capsule polysaccharides, and exopolysaccharides (Becker et al., 1995).
Ebosin, the first EPS reported in Streptomyces genus, is produced by Streptomyces sp. 139 (Wu et al., 1999). Previous studies showed that this EPS has antirheumatic arthritis activities in vivo (Zhang et al., 2013). A 27-gene cluster (ste) was identified as responsible for the biosynthesis of Ebosin (Wang et al., 2003), and the function of individual ste genes investigated (Sun et al., 2007; Bai et al., 2008; Qi et al., 2009; Li et al., 2010; Zhang et al., 2012). The present study focused on the function of ste9 gene that shows homology to some known bacterial chain length determinant (Wzz) proteins. We have confirmed that ste9 codes for a chain length determinant evidenced by its complementation to an E. coli wzz− mutant and plays an essential role in polymerization of the repeating units during Ebosin biosynthesis. To date, Wzz proteins have been studied mostly in Gram-negative bacteria (Franco et al., 1996; Kintz et al., 2008; Patrick et al., 2009; Sarnacki et al., 2009). In this study, Ste9 is characterized as a novel Wzz protein from the Gram-positive bacterium, Streptomyces.
Materials and methods
Bacterial strains and culture conditions
Streptomyces sp. 139 was isolated from a soil sample in China and kept in the China General Microbiology Culture Collection Center (No. 0405). The strain was cultured at 28 °C with shaking (250 r.p.m) in either TSB medium supplemented with 5 mM MgCl2 and 0.5% glycine or fermentation medium containing 1% glucose, 2% starch, 2% soybean extract, 0.2% tryptone, 0.2% beef extract, 0.4% yeast extract, 0.05% K2HPO4, 0.3% CaCO3, pH 7.3. Escherichia coli (E. coli) O86:H2 and O86:H2 (wzz−) strains were grown in LB medium at 37 °C.
DNA isolation and Southern analysis
Isolation of E. coli plasmid DNA and standard recombinant DNA techniques were performed as described by Sambrook & Russell (2001). Streptomyces plasmid and genomic DNA were isolated as mentioned by Kieser et al. (2000). For Southern analysis, a DIG high prime DNA labeling and detection starter kit II obtained from Roche was used according to the instructions of manufacturer.
Generation of deletion mutant
To evaluate the function of ste9 in Ebosin biosynthesis, knockout mutants were generated. A 1.093-kb internal fragment (F1, upstream of ste9) and another 1.114-kb internal fragment of ste9 (F2, downstream of ste9) were amplified by PCR from the chromosomal DNA of Streptomyces sp. 139 using primer pairs P1: 5′-TCAGAATTCGCAACATCATCAAGA-3′ and P2: 5′-CTCTCTAGACGTACTCGTCGTCATCG-3′ (EcoRI and XbaI restriction sites are underlined); P3: 5′-GGCTCTAGAGACCAGGAGGCGCG-3′ and P4: 5′-GGCAAGCTTCCGACAGCATCACCT-3′ (XbaI and HindIII restriction sites are underlined). The PCR amplification was performed under the following conditions: an initial denaturation at 98 °C for 2 min then 35 cycles of 45 s at 94 °C, 0.5 min at 59 °C, 2 min at 72 °C, and finally 10 min at 72 °C. A 1.23-kb fragment F3 carrying the Kmr gene was digested with XbaI from plasmid pUC19-Kmr (Zhang et al., 2006). The fragment containing F1, F3, and F2 was cloned into pKC1139 (Bierman et al., 1992) to create plasmid pKC9m. After propagation in E. coli ET12567 (MacNei et al., 1992), pKC9m was introduced into Streptomyces sp. 139 by polyethylene glycol (PEG)-mediated protoplast transformation (Kieser et al., 2000). Incubated at 28 °C for 16–20 h, the plates were overlaid with soft R2YE (0.7% agar) containing 40 μg mL−1 apramycin. Plasmid pKC9m bears a temperature-sensitive Streptomyces replication origin (MacNei et al., 1992) that is unable to replicate at temperatures above 34 °C. Therefore, the transformants were first incubated at 28 °C for 2 days until pinpoint-size colonies became visible and then shifted to 37 °C for further incubation. Mutants with Kmr Ams resulted from a double crossover via homologous recombination grew out of the original pinpoint-size colonies in several days. With a single crossover, the colonies with Kmr Amr could arise from the original conjugation.
Complementation of gene disruption mutants
With Streptomyces sp. 139 genomic DNA as template, the 1766-bp fragment of gene ste9 was amplified with PCR. The primers were P5: 5′-CTGTCTAGACCGGC- ATCGCGGC-3′ and P6: 5′-CCGAAGCTTGGTACGTGCCTGCGAT-3′ (XbaI and HindIII restriction sites are underlined). After joining the ermE* promoter and ste9 fragment, it was inserted into plasmid pKC1139 digested by EcoRI-HindIII to yield pKC9c. After propagation in E. coli ET12567 (pKC9c), pKC9c was isolated and protoplasts of Streptomyces sp. 139 (ste9−) transformed with this plasmid.
Isolation of Ebosin and its derivatives
Ebosin and its derivatives were isolated from the supernatants of fermentation cultures of Streptomyces sp. 139, mutant, and complement strains as described before (Chen et al., 2003).
Molecular exclusion chromatography
Molecular exclusion chromatography was performed to determine the peak molecular weight (Mp) of EPSs. The sample (10 mg) was dissolved in 1 mL of mobile phase (0.1 M Na2SO4) overnight at room temperature. Twenty microlitre of sample solution was injected into a TSK-GEL G-5000WXL column (7.8 × 300, Japan) with 0.1 M Na2SO4 as mobile phase and a flow rate of 0.4 mL min−1 at 25 °C (Waters 2414, Detector: 410, College park, MD).
Assay for IL-1R binding activity
The enzyme-linked immunosorbent assay (ELISA) method reported previously was used to analyze the binding activity for IL-1R of isolated EPSs (Zhang et al., 2012).
Cloning and expression of ste9 in E. coli O86:H2 (wzz−)
With Streptomyces sp. 139 genomic DNA as template, the ste9 gene coding region (1580 bp) was PCR-amplified using primers P7: 5′-CGGAATTCATGACGACG- AGTACGCC-3′ followed by P8: 5′-GCTAAGCTTCTACCGCGCCTCCTG-3′ (EcoRI and HindIII restriction sites are underlined) under the same conditions utilized for the amplification of fragments F1 and F2 mentioned above. The amplified DNA fragment was cloned into pET30a digested by EcoRI and HindIII to construct pET30a-ste9. The correct nucleotide sequence of ste9 gene fragment cloned into pET30a-ste9 was verified via sequencing using an ABI PRISM 377XL DNA Sequencer (Applied Biosystems). Competent E. coli O86:H2 (wzz−) cells were transformed with pET30a-ste9 and cultured overnight at 37 °C in LB broth containing 50 μg mL−1 kanamycin. The culture of E. coli O86:H2 (wzz−) (pET30a-ste9) was diluted to 1 : 20 with LB broth and subjected to further incubation at 37 °C; at the mean time, isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture at a final concentration of 1 mM. After 16 h of incubation at 37 °C, the bacteria were harvested and SDS-PAGE was performed to ascertain expression of ste9. While, with his-tag antibody as a probe, Western blot was also carried out (Sambrook & Russell, 2001).
LPS detection with silver stain
This protocol described by Marolda et al. (2006) was followed. After SDS-PAGE, the gel was soaked in 200 mL of fresh fixing solution (60% methanol and 10% acetic acid) overnight and washed for 30 min with 200 mL of 7.5% acetic acid. After discarding the acetic acid and adding 200 mL of 0.2% periodic acid, the gel was rocked for 30 min and then washed with deionized water for an hour changing the water every 15 min. Afterward, the gel was soaked in staining solution (42 mL of 0.36% NaOH, 2.8 mL of NH4OH concentrated solution, 8 mL of 19.4% AgNO3, and 148 mL of deionized water) prepared prior staining and rocked for 15 min. Washing with deionized water for 45 min and changing water every 15 min, the gel was soaked in developing solution (0.05% citric acid, 10% methanol, and 0.019% formaldehyde) and rocked until brown or yellow bands start to appear (5–15 min). With several changes of deionized water to stop developing, a photograph of the stained gel was taken as soon as possible.
Serum resistance assay
Following the method of Guo et al. (2005), serum resistance assays were performed with pooled normal human serum (Gemini Bio-product USA) and heat-inactivated serum, in which the concentration of serum was 80%. Heat inactivation was performed by incubating the serum at 56 °C for 30 min. A bacterial culture that had been allowed to grow overnight was diluted 1 : 100 in LB and grown to mid-log phase. The bacteria were then diluted 1 : 5 in pooled normal human serum and heat-inactivated serum and cultured at 37 °C. After 0, 1, 2, or 3 h, survival of the strains was tested by plating an aliquot on LB agar plates.
Data were showed as the mean ± SD from at least three independent experiments. The significance of differences between groups was evaluated by Student's t-test. P-values < 0.05 were considered significant.
Homology analysis of the Ste9
Database searches revealed that the deduced Ste9 protein bears 16.36% identity and 53.4% similarity, respectively, over 327-aa region to Wzz of Salmonella enterica (GenBank: CAA78946.1). 15.49% identity and 46.74% similarity separately over 368-aa region to Wzz of Shigella flexneri (GenBank: AAL50788) were showed also (Fig. 1a).
Kalynych et al. (2011) reported that Wzz proteins are inner membrane proteins with substantial variation in sequence identity (15–80%) but a conserved structural organization. In this report, the protein encoded by ste9 is homologous to some characterized Wzz protein originated from microorganism.
Disruption of ste9 and complementation of the knockout mutants
Three Kmr Ams colonies were selected randomly, and the isolated genomic DNA originated from Streptomyces sp. 139 (ste9−) and Streptomyces sp. 139 was digested with KpnI and SacI. The evidence of gene replacement of ste9 as shown in Fig. 1b was demonstrated by Southern hybridization using 1.114-kb DNA fragment (F2) probe (Fig. 1c). The hybridization signals appeared with the expected sizes of 2.754 kb (ste9− mutant) and 2.185 kb (wild-type strain). This result indicated that the colony with Kmr Ams phenotypes had integrated the kanamycin resistance cassette into the ste9 gene, which therefore has been disrupted.
Gene complementation of the knockout mutant was achieved by transforming the mutant cell with pKC9c and selection for apramycin resistance of transformants (Amr). Carriage of this plasmid was confirmed by restriction digestion mapping of the isolated plasmid by EcoRI-HindIII (not shown). The complemented strain was named Streptomyces sp. 139 (pKC9c).
Changes in molecular weight of EPS-9m and EPS-9c
With molecular exclusion chromatography, the peak molecular weight (Mp) of EPS-9m produced by Streptomyces sp. 139 (ste9−) was determined in comparison with Ebosin. The result showed that Mp of EPS-9m is 4.153 × 105 (Fig. 2b), which is remarkably smaller than that of Ebosin (9.033 × 105; Fig. 2a). The Mp of EPS-9c, product of the gene complementary strain Streptomyces sp. 139 (pKC9c), was 8.004 × 105, which was much higher than that of EPS-9m (Fig. 2c). These changes in molecular weights of Ebosin derivates indicate the involvement of ste9 in the chain length regulation during Ebosin biosynthesis.
The competitive binding activities of Ebosin derivates with IL-1 for IL-1R
Using an ELISA, the competitive binding activity of Ebosin derivates EPS-9m and EPS-9c with IL-1 for IL-1R were determined. The binding activities of EPS-9m for IL-1R were 22.51% (P < 0.01), 20.30% (P < 0.01), and 7.26% (P < 0.01),respectively (at 3.2, 0.64 and 0.128 ng μL−1), which were remarkably lower than those of Ebosin at the same concentrations. After gene ste9 complementation by plasmid transformation, the binding activities of EPS-9c increased to 30.42%, 28.45%, and 20.30% (P < 0.05), respectively (Fig. 3), which recovered partly from those of EPS-9m but lower than the original levels of Ebosin.
Expression of ste9 in E. coli O86:H2 (wzz−)
To test the assumed function of ste9 as a chain length determinant, the gene was cloned and expressed with pET30a as a vector in E. coli O86:H2 (wzz−). The SDS-PAGE of the cell lysate sample stained with Coomassie Blue demonstrated the recombinant protein with a molecular weight in agreement with the expected size of 60 kDa (including 2.5 kD of his•tag, originated from pET-30a; Fig. 4a), while Western blot showed the same band with his-tag antibodies as probe (Fig. 4b).
LPS of E. coli O86:H2 (wzz−) (pET30a-ste9)
Following gene expression of ste9 in E. coli O86:H2 (wzz−), the cell lysate sample was analyzed with SDS-PAGE and the LPS on the gel was detected with silver stain. While the intermediate modal distribution of LPS bands was lost in the mutant strain E. coli O86:H2 (wzz−), E. coli O86:H2 (wzz−) (pET30a-ste9) produced modal chain length LPS similar to that of the E. coli O86:H2 wild type (Fig. 5a). It indicated that Ste9 protein that originated from Streptomyces sp. 139 was functional as a Wzz protein in E. coli 86:H2 (wzz−).
Serum resistance of E. coli O86:H2 (wzz−) (pET30a-ste9)
O antigen (O polysaccharide) plays an important role in bacterial resistance to serum-mediated killing (Najdenski et al., 2003). Wzz protein is involved in the chain length regulation of a variety of polysaccharides including O antigens (Tang et al., 2007). Based on such dates, serum assays were performed with 80% serum and 80% heat-inactivated serum.
Escherichia coli O86:H2 (wzz−) was sensitive to the 80% human serum, exhibiting a decrease in viability of about 59.3% in the first hour and then 48% in the following 2 h compared with the wild-type strain. At the mean time, viable counts of the recombinant ste9-expressing strain reached similar levels to those of the wild strain after 2 h (Fig. 5b-1). Assays using the heat-inactivated serum demonstrated an even better restoration effect: the sensitivities between E. coli O86:H2 (wzz−) (pET30a-ste9) and E. coli O86:H2 were similar, but the decrease in viability of mutant strain E. coli O86:H2 (wzz−) was 20.3%, 13.3%, and 5.8% of those of the wild type at 1, 2, and 3 h, respectively (Fig. 5b-2).
Many microorganisms synthesize exopolysaccharides, which either remain attached to the cell surface or are found in extracellular medium. EPS includes a range of diverse polymers that play vital roles in a variety of biological processes. EPSs have found good applications in industry, including their use as biothickeners in foods. Notably, EPS produced by lactic acid bacteria (LAB) contributes significantly to the structure and viscosity of fermented milk products. Furthermore, several reports indicate that they can confer health benefits on consumers arising from their immunogenic and cholesterol-lowering properties (Boels et al., 2003). EPSs produced by Trichoderma erinaceum DG-312 had a strong anti-inflammatory activity against inflamed mice (Joo & Yun, 2005). Latiporus sulphureus var. miniatus produced EPSs with antidiabetic activity (Hwang et al., 2008).
Streptomyces is a group of Gram-positive bacteria that have been studied extensively, particularly with regard to their secondary metabolites. But little is known of the EPSs they produce. From Streptomyces sp. 139, we isolated a novel EPS called Ebosin, which has antirheumatic arthritis activities in vivo. To investigate its biosynthetic pathway, this paper reports on the role of ste9 gene, one of 27 ORFs present in the Ebosin biosynthesis cluster (Wang et al., 2003).
Database searches revealed that the deduced Ste9 protein bears homology to the Wzz domain of bacterial chain length determinant proteins. Reeves & Wang (2002) indicated that the function of Wzz is not strain specific in E. coli (Morona et al., 1994, 1995, 2000). To date, very little structure information is available for Wzz (Guo et al., 2006). Because the modality of O antigen can be easier observed by SDS-PAGE followed by silver staining, Wzz becomes the best model to study the mechanism of chain length of polysaccharide.
In this paper, SDS-PAGE analysis showed that E. coli O86:H2 (wzz−) (pET30a-ste9) produced a modal chain length lipid polysaccharide (LPS) similar to that of the wild-type E. coli O86:H2. At the same time, the expression of ste9 was able to restore the serum resistance of E. coli O86:H2 (wzz−) to almost the level of the wild-type strain. Utilizing the E. coli Wzz mutant strain O86:H2 (wzz−), it has been demonstrated that the ste9 gene is capable of restoring the function of Wzz and therefore confirmed the identity of this gene as coding for a chain length determinant in Streptomyces sp. 139.
Disruption of ste9 dramatically affected the biosynthesis of Ebosin as evidenced by the significant reduction in molecular weight of EPS (EPS-9m), which can then be partially restored after complementation. It was attributed to the reduction of polymerization caused by the loss of Ste9 protein. This failure in proper polymerization also resulted in the loss of bioactivity of the EPS for binding to IL-1R. Such results indicate that the ste9 gene codes for a chain length determinant which plays an essential role in Ebosin biosynthesis.
Wzz proteins have been studied mostly in Gram-negative bacteria. Sergei et al. reported that Gram-negative bacteria usually have two different Wzz proteins, which confer two distinct OAg model chain lengths, one longer and other shorter (Kalynych et al., 2011). Different Wzz proteins confer a wide range of modal lengths (4 to > 100 repeat units). Study showed that O antigen of 16–35 repeat units was found to activate complement more efficiently than other length during heterologous wzz gene expression (Gerald et al., 2006). At present, the reports about Wzz in Gram-positive bacteria have not been found. We wonder that whether Wzz proteins in Gram-positive bacteria confer a wide range of modal lengths also.
This research was supported by a grant from the Natural Science Foundation of China (NSFC 30530830). It was also supported by grants from the National Key Project of New Drug Study of China (2010ZX09401-403, 2012ZX09301002-001-023-02).