Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Identification and Functional Analysis of the Chain Length Determinant Gene ste8 Involved in the Biosynthesis of Ebosin by Streptomyces sp. 139

  • Yang, Zhang (Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College) ;
  • Li, Xiaohua (Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College) ;
  • Qi, Xiaoqaing (Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College) ;
  • Shan, Junjie (Institute of Pharmacology and Toxicology) ;
  • Jiang, Rong (Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College) ;
  • Guo, Lianhong (Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College) ;
  • Zhang, Ren (School of Biological Sciences, University of Wollongong) ;
  • Li, Yuan (Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College)
  • Received : 2013.05.15
  • Accepted : 2013.07.11
  • Published : 2013.11.28
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Abstract

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has obvious antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste), consisting of 27 open reading frames, has been identified. This paper reports our study of the gene functionality of ste8, the predicted protein product of which is homologous to some bacterial chain length determinant Wzz proteins. For characterization of Ste8, ste8 was cloned and expressed in the mutant strain E. coli 086:H2 (${\Delta}wzz$). The functional complementation of wzz by ste8 was demonstrated by the restoration of wild-type lipopolysaccharide biosynthesis and increased levels of serum resistance of E. coli 086:H2 (${\Delta}wzz$) (pET30a-ste8). To examine the function of ste8 in ebosin biosynthesis, the gene was knocked out with a double crossover via homologous recombination. The molecular weight of the ebosin derivative EPS-8m produced by the mutant Streptomyces sp. 139 ($ste8^-$) was much lower than that of ebosin, and the binding activity of EPS-8m for IL-1R decreased significantly compared with ebosin. These results demonstrate that ste8 encodes a chain length determinant (Wzz) that functions in ebosin biosynthesis.

Keywords

Introduction

Lipopolysaccharides (LPS), one of the cell surface polysaccharides, consist of three structural parts: lipid A, core oligosaccharide, and O-antigen. The O-antigen plays an important role in bacterial resistance to serum-mediated killing [21]. During the biosynthesis of heteropolymeric Oantigen, the chain length determinant protein Wzz is proposed to terminate polymerization and regulate polysaccharide chain length. Wzz proteins are 36-40 kDa inner membrane proteins, the primary sequences of which are very well conserved, as are the predicted membrane topologies [24,26]. Such proteins are characterized by two transmembrane segments located in the amino-terminal and carboxyterminal, with a large hydrophilic loop located in the periplasm [15]. It seems that amino acids throughout the whole region of the Wzz protein are involved in regulation [15]. As a member of the“polysaccharide co-polymerase” superfamily, Wzz proteins are involved in the regulation of chain length of a variety of polysaccharides, including Oantigens, capsular polysaccharides and exopolysaccharides (EPSs) [26].

The biosynthesis gene cluster (ste) of e b osin, a n ovel exopolysaccharide produced by Streptomyces sp. 139, consists of 27 ORFs [27]. Some of the genes involved in the cluster have been studied separately [5,16,29]. In this paper, a functional study of ste8 is presented. According to the database, Ste8 is homologous with the domain of the chain length determinant (Wzz) originating from some microbes. For characterization of Ste8, ste8 was cloned and expressed in the mutant strain Escherichia coli 086:H2 (Δwzz). According to an analysis of the LPS produced by E. coli 086:H2 (Δwzz) (pET30a-ste8) and the serum resistance of the recombinant strain, the complementary function of gene ste8 has been identified for gene wzz in the mutant strain E. coli 086:H2 (Δwzz). After disruption of ste8 in Streptomyces sp. 139, the molecular weight and binding activity with IL-1R of EPS- 8m produced by the mutant strain Streptomyces sp. 139 (ste8-) decreased drastically compared with ebosin. These results demonstrate that ste8 encodes a chain length determinant (Wzz) and plays an important role in ebosin biosynthesis.

 

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℃ with shaking (418 g) in either TSB medium supplemented with 5 mM MgCl2 and 0.5% (w/v) 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, and 0.3% (w/v) CaCO3, (pH 7.3). E. coli 086:H2 and 086:H2 (Δwzz) were grown at 37℃ in Luria-Bertani (LB) medium.

DNA Preparation and Southern Blot Analysis

Standard recombinant DNA techniques were performed as described by Sambrook and Russell [23]. Streptomyces plasmid and genomic DNA were isolated as described by Kieser et al. [14]. For Southern blot analysis, a DIG High Prime DNA Labeling and Detection Starter Kit II obtained from Roche (USA) was used according to the manufacturer’s instructions.

Table 1.Bacterial strains and plasmids used in this study.

Cloning and Expression of ste8 in E. coli 086:H2 (Δwzz)

With Streptomyces sp. 139 genomic DNA as the template, ste8 was amplified by PCR using the primers P1: 5’-CAGAATTCATGTTCCGCCGACGC-3’ and P2: 5’ TACAAGCTTTCATCGCGTACCTCCCC-3’ (EcoRI and HindIII restriction sites are underlined, respectively). PCR amplification was performed under the following conditions: an initial denaturation at 98℃ for 2 min; then 30 cycles of 45 sec at 98℃, 30sec at 59℃, 2 min at 72℃, and finally 10m in at 72℃. A 1,346 bp amplified ste8 DNA fragment was cloned into the plasmid pET30a digested with EcoRI-HindIII to construct the recombinant plasmid pET30a-ste8, which was then transformed into E. coli 086:H2 (Δwzz) competent cells. Three transformants were selected and cultured overnight at 37℃ in LB broth containing kanamycin (50 μg/ml). The E. coli 086:H2 (Δwzz) (pET30a-ste8) culture was diluted to 1:20 with LB broth and subjected to further incubation at 37℃, and isopropyl-β-thiogalactopyranoside was added to the culture at a final concentration of 1 mM. After incubation for 16 h at 37℃, the bacteria were harvested and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to ascertain the expression of ste8. Meanwhile, western blot analysis was also carried out using His-tag antibody as the probe [23].

LPS Detection with Silver Stain

According to the protocol described by Marolda et al. [18], an LPS detection assay was performed. After SDS-PAGE, the gel was soaked in 200 ml of fresh fixing solution (60% methanol and 10% acetic acid) overnight and washed for 30m in with 200 ml of 7.5% acetic acid. After removal of the acetic acid and addition of 200 ml of 0.2% periodic acid, the gel was rocked for 30 min, washed with deionized water for 1 h, and the water changed every 15 min. Then, the gel was soaked in staining solution (w/v, 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 to staining, and rocked for 15 min. After washing with deionized water for 45 min and changing the 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 began to appear (5-15 min). After several changes of deionized water to stop color developing, a photo of the stained gel was taken as soon as possible.

Serum Resistance Assay

Using the method of Guo et al. [9], serum resistance assays were carried out with pooled normal human serum (Gemini Bio- Products, USA) and heat-inactivated serum, in which the serum concentration was 80%. Heat inactivation was performed by incubating the serum at 56℃ for 30min. A bacterial culture that had been allowed to grow overnight was diluted 1:100 with LB and grown to mid-log phase. The bacteria were then diluted 1:5 in pooled normal human serum or heat-inactivated serum and cultured at 37℃. After 0, 1, 2, or 3 h, survival of the strains was tested by plating an aliquot on LB agar plates.

Disruption of ste8

A 748 bp fragment (F1) upstream of ste8 was PCR amplified with primers P3: 5’-CTGAATTCCCCATCTGCTCTGCTACC-3’ and P4: 5’ TGTCTAGACGGGAACACCAGGGAGG-3’ (EcoRI and XbaI restriction sites are underlined, respectively). Another 762 bp fragment (F2) downstream of ste8 was also PCR amplified using primersP5: 5’-GCTTCTAGAGGTACGCCATGACGACG-3’ and P6: 5’ CTTAAGCTTGGGCCTTGGGCATCGAG-3’ (XbaI and HindIII restriction sites are underlined, respectively). The conditions of PCR amplification were the same as mentioned above. A 1.2 kb fragment, F3, carrying the Kmr gene, was digested with XbaI from plasmid pFD666 [7]. The fragment containing F1, F3, and F2 was cloned into pKC1139 [2] to construct plasmid pKC8m. After propagation in E. coli ET12567 [17], pKC8m was introduced into Streptomyces sp. 139 by polyethyleneglycol-mediated protoplast transformation [14]. After incubation at 28℃ for 16-20h, the plates were overlaid with soft R2YE (0.7 % agar )undefined(w/v)(40 μg/ml). Plasmid pKC8m bears a temperaturesensitive Streptomyces replication origin [7] that is unable to replicate at temperatures above 34℃. Therefore, the transformants were first incubated at 28℃ for 2 days until pinpoint-sized colonies became visible and then were shifted to 37℃ for further incubation. Mutants resulting from a double crossover via homologous recombination grew out of the original pinpoint-sized colonies in several days.

Complementation of ste8 Disruption Mutant

Using the genomic DNA of Streptomyces sp. 139 as a template, a 1,413 bp ste8 fragment was amplified by PCR with the primers P7: 5’ GCGTCTAGAGCACGTCCCTTGAGCG-3’ and P8: 5’-CGTAAGCTTGGCCGGCGTACTCGTC-3’ (XbaI and HindIII restriction sites are underlined, respectively). The 0.45 kb fragment of the ermE* promoter was isolated from the plasmid pGEM-3zf-ermE* [1] digested with EcoRI-XbaI. After ligating the ermE* fragment and the 1,413 bp ste8 fragment, the resulting fragment was inserted into plasmid pKC1139 digested with EcoRI-HindIII to create pKC8c, which was then transformed into E. coli ET12567. After cultivation of the recombinant strain with Amr (50 μg/ml), the plasmid pKC8c was isolated and transformed into the protoplasts of Streptomyces sp. 139 (ste8-). The complementing strain was designated as Streptomyces sp. 139 (pKC8c).

Molecular Exclusion Chromatography

To determine the peak molecular weight (Mp) of EPSs, molecular exclusion chromatography was performed. The sample (10mg) was dissolved in 1 ml of mobile phase (0.1 M Na2SO4) overnight at room temperature. Twenty microliters of sample solution was injected into a TSK-GEL G-5000WXL column (7.8 × 300; Japan) with 0.1 M Na2SO4 as the mobile phase and a flow rate of 0.4 ml per minute at 25℃ (Waters 2414, Detector: 410; College Park, MD, USA).

Isolation of EPSs

Ebosin and its derivatives (EPS-8m, EPS-8c) were isolated separately from their producing strains according to a previously described protocol [6].

Assay for IL-1R Binding Activity of EPSs

A previously reported enzyme-linked immunosorbent assay (ELISA) method was used to analyze the IL-1R binding activity of isolated EPSs [29].

Statistical Analysis

Data are shown 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 less than 0.05 were considered significant.

 

Results

Homology Between Ste8 and the Chain Length Determinant (Wzz) Originating from Microorganisms

The DNA sequence of ste8 presented in this study was deposited in GenBank under Accession No. AY131229. Database searches revealed that the deduced Ste8 protein bears 16.62% identity and 49.05% similarity to a 368 aa region of Wzz of Shigella sonnei (GenBank Accession No. AAG17417.1). In addition, the deduced sequence shares 15.15% identity and 50.22% similarity to a 231 aa region of Cps19aC of Streptococcus pneumonia (GenBank Accession No. AAC78665.1) (Fig. 1). The deduced Ste8 protein also bears 9.17% identity and 49.77% similarity to a 415 aa region of Wzz of E. coli 086:H2 (GenBank Accession No. AY667408.1).

Fig. 1.Homology between Ste8 and the chain length determinant (Wzz) of microbial origin.

Kalynych et al. [13] reported that Wzz proteins are inner membrane proteins with substantial variation in sequence identity (15-80%) but a conserved structural organization. In this paper, the results of homology analysis showed that the deduced Ste8 sequence was generally in agreement with that of the expected Wzz proteins.

Cloning and Expression of ste8 in E. coli 086:H2 (Δwzz)

The ste8 gene was amplified by PCR from Streptomyces sp. 139 genomic DNA and cloned into pET30a, which was then expressed in the mutant strain E. coli 086:H2 (Δwzz) by transformation. SDS-PAGE of cell lysate samples stained with Coomassie blue showed the recombinant protein with a molecular mass in agreement with the expected size, ~54 kDa (Fig. 2A). This protein band was verified by western blot analysis using the His-tag antibody as a probe (Fig. 2B).

Fig. 2.SDS-PAGE (A) and western blot (B) analyses of lysates of E. coli 086:H2, E. coli 086:H2 (Δwzz), and E. coli 086:H2 (pET30a-ste8). (A) 1, E. coli 086:H2; 2, E. coli 086:H2 (Δwzz); 3, Protein size markers; 4, E. coli 086:H2 (Δwzz) (pET30a-ste8). (B) 1, E. coli 086:H2 (Δwzz); 2, E. coli 086:H2 (Δwzz) (pET30a-ste8).

Fig. 3.SDS-PAGE analysis of wild-type and mutant E. coli 086:H2 LPSs. 1, Lysate of wild-type strain E. coli 086:H2; 2, Lysate of mutant strain E. coli 086:H2 (Δwzz); 3, Lysate of recombinant strain E. coli 086:H2 (Δwzz) (pET30a-ste8).

LPS Analysis of E. coli 086:H2 (Δwzz) (pET30a-ste8)

Lysates of E. coli 086:H2 (Δwzz) (pET30a-ste8), E. coli 086:H2 (Δwzz), and E. coli 086:H2 were analyzed by SDS-PAGE followed by silver staining. LPS of the E. coli 086:H2 wild-type strain had an intermediate modal distribution of bands (10-18 O units), which were not observed in the sample from the mutant strain. Meanwhile, LPS produced by the recombinant strain E. coli 086:H2 (Δwzz) (pET30a-ste8) also exhibited an intermediate modal distribution of bands, similar to that of the wild-type strain (Fig. 3). These results demonstrate the restoration of wzz gene function in the mutant strain E. coli 086:H2 (Δwzz) by the Streptomyces ste8 gene.

Serum Resistance of E. coli 086:H2 (Δwzz) (pET30a-ste8)

As Guo et al. [9] described, E. coli 086:H2, which contains relatively long O chains, is serum resistant. In this study, assays were performed with 80% serum and 80% heatinactivated serum. Strains at the exponential phase of growth were added to serum and cultivated for 3 h. Viable counts were determined in duplicate at the beginning of the experiment and then every hour for 3 h.

As shown in Fig. 4A, wild-type E. coli 086:H2 was resistant to 80% serum, with viable counts increasing to ~300% in the first hour and then reaching over 400% an hour later. The viable counts of mutant strain E. coli 086:H2 (Δwzz) were lower than those of the wild-type strain, about 59.3% at the first hour and then 48% in the next 2 h. At the same time, the viability of E. coli 086:H2 (Δwzz) (pET30a-ste8) was much higher than that of the mutant, almost reaching the levels of the wild type at hours 2 and 3. Assays using the heat-inactivated 80% serum demonstrated that E. coli 086:H2 and E. coli 086:H2 (Δwzz) (pET30a-ste8) grew nearly equally, but the decrease in viability of the mutant strain E. coli 086:H2 (Δwzz) was 16.9%, 13.3%, and 5.8% at 1, 2, and 3 h, respectively, compared with that of the wildtype strain (Fig. 4B). These results provide further evidence that ste8 can complement the function of wzz in E. coli 086:H2 (Δwzz).

Fig. 4.Serum assay using 80% serum (A) and heat-inactivated serum (B). The assays were performed for 3 h. Samples were taken in duplicate and the percentage of surviving cells was plotted against incubation time.

Disruption of ste8 and Complementation of the Mutant

To study the function of ste8 in relation to ebosin biosynthesis, the gene was disrupted (Fig. 5A). Two colonies (Kmr Ams ) were randomly selected. Genomic DNAs of the mutant strain (ste8-) and wild-type strain were digested separately with BamHI, and then subjected to agarose gel electrophoresis. Southern hybridization was carried out with a 672 bp upstream fragment of ste8 as a probe. As shown in Fig. 5B, a distinctive hybridization band of 3.40 kb was detected in the mutant strain Streptomyces sp. 139 (ste8-), and a 1.38 kb band appeared in the wild-type strain as predicted. Hence, the kanamycin resistance cassette was confirmed to have been integrated into ste8 in the strains with Kmr Ams, which therefore lost the gene function of ste8.

Fig. 5.Confirmation of ste8 gene disruption. (A) Diagram of ste8 gene replacement with a double crossover via homologous recombination. The gray box indicates the location of ste8. Restriction maps of wild-type Streptomyces sp. 139 and the mutant (ste8-) show the predicted fragment sizes upon BamH1 digestion. (B) Southern blot autoradiograph of wild-type and knockout mutant Streptomyces sp. 139 (ste8-) strains. 1, Chromosomal DNA of Streptomyces sp. 139 digested with BamHI; 2, chromosomal DNA of knockout mutant Streptomyces sp. 139 (ste8-) digested with BamHI.

Gene complementation of the knockout mutant was achieved by transforming the mutant cells with pKC8c. It was evidenced by the apramycin resistance of the transformants (Amr) and correct restriction mapping of the isolated plasmid by EcoRI-HindIII (data not shown).

Determination of the Molecular Weight of EPSs

The peak molecular weights (Mps) of EPS-8m, EPS-8c and ebosin were determined by molecular exclusion chromatography. EPS-8m isolated from the mutant strain Streptomyces sp. 139 (ste8-) had two components with Mps of 40.02 × 104 and 2.57×104 (Fig. 6B), which are remarkably lower than that of ebosin (90.33 × 104) (Fig. 6A). After gene complementation, EPS-8c produced by the complementing strain Streptomyces sp. 139 (pKC8c) exhibited an Mp of 40.21 × 104, similar to that of the EPS-8m large component, whereas the component with an Mp of 2.57 × 104 disappeared, showing partial restoration of the Mw of EPS-8c compared with EPS-8m.

Competitive Binding Activity of Ebosin Derivatives with IL-1 for IL-1R

Using an ELISA assay, the competitive binding activity of the ebosin derivatives EPS-8m and EPS-8c with IL-1 for IL-1R were determined. The binding activities of EPS-8m were 19.86% (P < 0.05), 10.31% (P < 0.05), and 0.28% (P < 0.001) at dosages of 3.2, 0.64, and 0.128 ng/μl, respectively, which were obviously lower than those of ebosin at 54.28%, 50.12%, and 41.59% at the same concentrations. The activities of EPS-8c were 31.17% (P < 0.05), 28.59% (P < 0.05), and 11.16% (P < 0.05) at concentrations of 3.2, 0.64, and 0.128 ng/μl, respectively, which were partially restored compared with EPS-8m but still lower than those of ebosin (Fig. 7).

 

Discussion

Exopolysaccharides are long-chain polysaccharides consisting of branched, repeating units of sugar or sugar derivatives. The biosynthesis of EPSs starts with the intracellular formation of EPS precursors, the sugar nucleotides, followed by the formation of repeating units on a lipid carrier located in the cytoplasmic membrane. The later step involves transport of the repeating units across the membrane to the outer layer and polymerization of numerous units to form EPSs [4]. EPSs have been widely used in the food industry [25] and were recently found to have some health benefits such as cholesterol-lowering properties [3], anti-inflammatory activity [12], and antidiabetic activity [11].

Fig. 6.Molecular exclusion chromatograms of (A) ebosin, (B) EPS-8m, and (C) EPS-8c produced by Streptomyces sp. 139, knockout mutant Streptomyces sp. 139 (ste8-), and Streptomyces sp. 139 (pKC8c), respectively.

All lipopolysaccharides consist of three regions: O-antigen polysaccharide, core oligosaccharide, and lipid A. The chain length determinant protein Wzz is important for determining the length of the O-antigen side chain attached to LPS, which regulates the number of subunits added together, giving rise to a narrow strain-specific range of O-antigen chain lengths [8,19]. To date, Wzz proteins have been studied mostly in Gram-negative bacteria [8,15,22,24].

Gram-positive Streptomyces bacteria form an important group of industrial microorganisms. Previous studies in our laboratory showed that ebosin is a novel EPS isolated from Streptomyces sp. 139, having antirheumatic arthritis activity in vivo [28]. The ebosin biosynthesis gene cluster (ste) consists of 27 ORFs [27]. We have endeavored to investigate the function and potential use of these ste genes.

Fig. 7.Binding activity of ebosin, EPS-8m, and EPS-8c for IL-1R at 3.2, 0.64, and 0.128 ng/μl, respectively, by ELISA

In this paper, we dealt with the gene ste8, located on the ste cluster, for functional characterization. Based on an analysis of protein sequence homology, the hypothesis was that ste8 codes for a chain length determinant Wzz protein. Two lines of evidence presented herein support this hypothesis. Firstly, ste8 complemented the function of the knocked-out wzz gene in the mutant strain E. coli 086:H2 (Δwzz), restoring its ability to produce LPS as the wild-type strain does and the serum resistance of E. coli 086:H2 (Δwzz) to almost the level of the wild-type strain. Therefore, we confirmed that this gene encodes a chain length determinant in Streptomyces sp. 139. Secondly, after disruption of ste8 in Streptomyces sp. 139, the polymerization of EPS (ebosin) during biosynthesis was dramatically reduced along with the bioactivity of mutant EPS-8m. In short, ste8 is functional in Streptomyces sp. 139 as is the gene wzz encoding the chain length determinant Wzz in other bacteria, and it undoubtedly plays an essential role during ebosin biosynthesis.

To date, Wzz proteins have been studied mostly in Gramnegative bacteria [15,22,24]. Gram-negative bacteria often have two different Wzz proteins, which confer two distinct O-antigen model chain lengths, one longer and the other shorter [13]. Different Wzz proteins confer a wide range of modal lengths (4 to >100 repeat units) [10]. Murray et al. [20] demonstrated that the wzz mutant in one species of bacteria can be complemented by expression of the wzz gene from another bacterium. Results showed that altering the length of the lipopolysaccharide O-antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with complement. O-antigen of 16-35 repeat units was found to activate complement more efficiently than other lengths. Wzz in Gram-positive bacteria has not been reported. We wonder if Wzz proteins in Grampositive bacteria also confer a wide range of modal lengths. Although there is no experimental evidence, Ste8 may be speculated to confer a shorter model length, which cannot activate complement more efficiently.

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