Keywords

1 Introduction

A number of bacteria of the genus Sphingomonas produce polysaccharides called sphingans, including gellan, welan, S-88, rhamsan, and diutan [1–3]. Sphingans share the similar tetrasaccharide backbone structures and divergent side chains. Because of their excellent rheological characteristics, sphingans have been utilized for a wide range of biotechnological applications in the food, oilfield, and pharmaceutical industries [4–7]. In recent years, with the continuous exploration of microbial resources, some new sphingan-secreting strains have been isolated from diverse environments [8]. Sphingomonas sanxanigenens strain NX02 is a new species of the genus Sphingomonas sensu stricto that was isolated from soil [9]. Strain NX02 synthesizes a novel sphingan called sphingan Ss, with a linear tetrasaccharide repeat unit consisting of glucose, glucuronic acid, rhamnose, and mannose [10]. Although sphingan Ss has been used in the field of oil exploitation, its mechanism of synthesis is still unknown.

The complete biosynthetic pathway of gellan, S-88, and diutan are presently known. It is a multistep process that can be divided into three sequential steps: intracellular synthesis of the nucleotide-sugar precursors, assembly of the tetrasaccharide repeat units linked to the inner membrane, and translocation of the repeat units to the periplasmic space followed by their polymerization and export through the outer membrane [11–13]. Polymerase, encoded by the spsC gene, catalyzes the tetrasaccharide repeat units to polysaccharide. The spsC protein involves in sphingan polysaccharide chain length determination [14, 15].

In this paper, a mini-Tn5 transposon mutant strain of S. sanxanigenens NX02, which cannot produce sphingan Ss, was screened and isolated. The complete ORF sequence of spsC gene was obtained by TAIL PCR. The phylogenetic relation and protein characteristic was analyzed with bioinformatics method.

2 Materials and Methods

2.1 Bacterial Strains, Plasmids, and Growth Conditions

Escherichia coli strains DH5a (Transgen, Beijing, China) were used as host cells for gene cloning. E. coli strains S17-1(mini-Tn5) were used as donor strains for transposon mutagenesis. S. sanxanigenens strain NX02 was cultured on NK medium (15 g glucose l−1, 5 g peptone l−1, 3 g beef powder l−1, 1 g yeast extract l−1, and 15 g agar l−1, pH 7.0) at 30 °C. The fermentation medium contained the following: 45 g glucose l−1, 2.5 g NaNO3 l−1, 0.2 g yeast extract l−1, 1.2 g K2HPO4 l−1, 1 g CaCO3 l−1, 0.005 g FeSO4 l−1, 0.4 g NaCl l−1, and 1 g MgSO4 l−1, pH 7.5. pEASY-Blunt (Transgen) was employed as gene cloning. When required, the culture medium was supplemented with 100 mg ampicillin l−1, 30 mg chloramphenicol l−1, or 10 mg kanamycin l−1. Peptone, beef powder, yeast extract, agar, and other chemicals were purchased from Dingguo Limited (Tianjin, China).

2.2 Transposon Mutagenesis

Suicide plasmid with transposon mini-Tn5 was transferred from donor strain E. coli S17-1 into recipient strain S. sanxanigenens NX02 by mobilization with a filter mating technique [16]. E. coli S17-1(mini-Tn5) was incubated for 12 h at 37 °C with 10 mg kanamycin l−1, and S. sanxanigenens NX02 was incubated for 24 h at 30 °C with 30 mg chloramphenicol l−1. Filters with the mixture of donor and recipient strains in a 1:4 ratio were incubated for 8 h at 30 °C on the surface of NK medium plates. Cells were then suspended in 10 mM MgSO4, and the appropriate dilutions were plated on selective medium with kanamycin and chloramphenicol. The mini-Tn5 transposon mutant strains were screened by the viscous phenotype of colony.

2.3 DNA Techniques

Standard procedures, including DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, DNA ligation, transformation of E. coli, and S. sanxanigenens, were performed using conventional methods [17]. Genomic DNA was extracted by LiCl precipitation [18]. Plasmid DNA was purified from E. coli by the alkaline lysis procedure or using the AxyprepTM Plasmid Miniprep Kit [19].

2.4 Cloning and Sequence Determination of spsC Gene

The flanking sequences of mini-Tn5 transposon insertion site was obtained by the method of Tn5 external direction PCR amplification and long primer RAPD, using the following primers: Wt1 (5'- CAATAGCGTTATCAACCCGCT-3'), Wt2 (5'-CCAAACGTTGACACCCAGTT-3'), Ric1(5'-ATGTAAGCTCCTGGGGATTCAC-3'), Ric2(5'-AAGTAAGTGACTGGGGTGAGCG-3'), Box(5'-CTACGGCAAGGCGACGCTGACG-3'), Rep1(5'-IIIICGICGICATCIGGC-3'), Rep2 (5'-ICGIC TTATCIGGCCTAC-3'). The PCR product was sequenced, and analysis of the deduced amino acid sequence confirmed that it contained an incomplete open reading frame (ORF) and that the deduced amino acid sequence was homologous to GelC protein sequences in data banks. The complete ORF sequence of spsC was obtained by thermal asymmetric interlaced (TAIL) PCR.

2.5 Sequence Alignment and Bioinformatics Analysis

Sequence similarity searches were performed using BLAST 2.0 [20] at NCBI. Alignments to determine protein and DNA similarities were performed using the CLUSTAL method [21] and a phylogenetic tree was constructed using MEGA 4.0 [36] with the neighbor-joining method [22]. Sequence data were analyzed with DNAMAN 5.0 (Lynnon Biosoft, Quebec, Canada). The physicochemical and hydrophobic properties of protein spsC were obtained with program ProtParam and ProtScale, respectively. The protein secondary structure prediction was analyzed with program PSIPRED [23].

3 Results and Discussion

3.1 Tn5-Induced Sphingan Ss-Deficient Mutants of S. sanxanigenens NX02

A library of random mini-Tn5 insertions was constructed in S. sanxanigenens NX02 as described in the experimental section. Colonies were individually screened for sphingan Ss deficient at NK medium plate with chloramphenicol and kanamycin. The morphological character of wide-type strain NX02 was convex and viscous (Fig. 1.1a). The flat and tenuous colony of mutant T163 indicated that sphingans Ss was not secreted from the mutant (Fig. 1.1b). This result was then confirmed by shake flask fermentation experiment. The result of PCR showed that the phenotypic change of mutant T163 was caused by mini-Tn5 insertion.

Fig. 1.1
figure 1

Colony characteristics of strain NX02 and transposon mutant T163

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3.2 Cloning of spsC Complete ORF Sequence

The flanking sequences of mini-Tn5 insertion site were amplified by PCR. As shown in Fig. 1.2, two electrophoretic bands of about 1,200 and 1,100 bp were obtained (lane 1–2). With DNA sequencing and TAIL PCR, the complete ORF sequence of spsC was obtained as shown in lane 3. The nucleotide sequence of spsC gene has been deposited in the GenBank database under the accession number AGQ04616.

Fig. 1.2
figure 2

PCR result of spsC gene

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Nucleotide sequencing of spsC in S. sanxanigenens revealed a unique 1,482-nt ORF, starting with a putative ATG start codon. Preceding the start codon (8 nt upstream), a putative ribosome-binding site (RBS) (5'-GGGGA-3') was identified by taking into consideration previous descriptions of RBSs from S. paucimobilis ATCC31461 [14]. However, typical −10 and −35 regions were not identified upstream of the predicted Shine-Dalgarno (SD) sequence. The spsC gene has a high G +C content (68 %) and a high frequency of G or C in the third position (87 %), which is characteristic of Sphingomonas genes [24] and consistent with that of S. sanxanigenens [25].

3.3 Phylogenetic Analysis of spsC Amino Acid Sequence

The putative amino acid sequence encoded by the spsC was compared with data deposited in the GenBank database. The following high levels of identity with other proteins from a variety of organisms were detected: 60 % identity with polysaccharide biosynthesis protein of Novosphingobium lindaniclasticum LE124 (EQB15321) and 58 % identity with Sphingomonas sp. LH128 (EJU14361), followed by 55 % identity with Novosphingobium sp. AP12 (EJL23329), 52 and 51 % identity with protein from Sphingomonas sp. PR090111-T3T-6A (WP_019832308) and Sphingobium sp. YL23 (WP_022681617). Construction of a phylogenetic tree for the spsC proteins (Fig. 1.3) revealed two obviously divergent phylogenetic groups of prokaryotes. spsC of S. sanxanigenens was in the group including protein of N. lindaniclasticumand LE124 and Sphingomonas sp. LH128, but further apart from the group including protein of Sphingopyxis baekryungensis and Sphingomonas wittichii RW1. Homologous analysis showed that the most sequence of spsC had high identity with GumC protein which involved in exopolysaccharide Xanthan biosynthesis, and the N-terminal sequence of spsC had high identity to chain length determinant protein of Wzz superfamily.

Fig. 1.3
figure 3

Phylognetic tree of spsC amino acid sequence

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3.4 Properties of Protein spsC from S. sanxanigenens

The protein spsC gene is composed of 493 amino acids, with a calculated molecular mass of 53.51 kDa and a predicted isoelectric point (PI) of 9.42. Analysis of the amino acid composition of spsC revealed a composition of 54 % polar residues and 46 % hydrophobic residues. The amount of basic and acidic residues was 66 and 55. The result of hydrophobic analysis showed that the aliphatic index of spsC was 0.93, and the instability index was computed to be 52.49 (Fig. 1.4). The analysis of PSIPRED showed that spsC have two transmembrane domains flanking a central extracellular segment. The determination of length distribution of the polysaccharide chains is controlled by a family of proteins termed polysaccharide polymerases (PCP). PCP enzymes involved in extracellular polysaccharides synthesis systems in Gram-negative bacteria have, in addition to the membrane/periplasmic domain, a cytoplasmic domain of protein tyrosine kinases, and the prototype of this family is Wzc from Escherichia coli [26]. The PCP enzyme in S. sanxanigenens NX02 is encoded by the gene spsC. The hydrophobic plot for spsC suggested the presence of two putative transmembrane α-helices, located between amino acids Y13-V44 and P411-L436, respectively. The protein of spsC shows the typical PCP N- and C-terminal transmembrane helices separated by a segment with a predicted coil region located in the periplasm.

Fig. 1.4
figure 4

Hydrophobicity and hydrophilicity analysis of spsC

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4 Conclusion

Sphingomonas sanxanigenens NX02 is a new species of the genus Sphingomonas and has low homology with other known sphingan producing strains. The complete ORF sequence of sphingan gene cannot be obtained by standard PCR with degenerate primers. Screening deficient mutants is the necessary way to obtain the gene information about sphingan Ss. In this study, the complete ORF sequence of spsC gene from S. sanxanigenens was cloned and characterized for the first time. Bioinformatics analysis showed that the sequence of spsC had high identity with GumC protein and chain length determinant protein of Wzz superfamily. The protein of spsC showed the typical polysaccharide polymerases family transmembrane helices and periplasm coil region. This work should prove useful for further research into sphingan Ss synthesis pathways and genetic engineering with a view to control sphingan Ss production.