Distribution of Sorangium cellulosum

The myxobacterium Sorangium cellulosum exists and plays an important role in soil ecology by its ability to degrade cellulosic materials; it was found to be widely distributed in North American soils.     

Cloning and characterization of an rRNA methyltransferase from Sorangium cellulosum

A locus (kmr) responsible for aminoglycosides-resistance of Sorangium cellulosum was cloned and characterized in Myxococcus xanthus. The gene kmr encodes a putative rRNA methyltransferase. Expression of the complete ORF endowed the Myxococcus transformants with the resistance to aminoglycosidic antibiotics of kanamycin, apramycin, gentamycin, neomycin, and tobramycin at an extraordinary high-level (MIC, higher than 500 μg/ml). However, the gene did not function in Escherichia coli cells. In Sorangium genome, the gene kmr was followed by a putative integrase gene, and was highly homologous in different Sorangium strains. The Sorangium rRNA methyltransferase sequence was in low similarity to the reported 16S rRNA methyltransferases, and their resistance spectrums were also different. The results indicate that the rRNA methyltransferase (Kmr) in Sorangium strains is a new member of the rRNA methyltransferases family.     

Repeated batch production of epothilone B by immobilized Sorangium cellulosum

Production of extracellular epothilone B, one of the potent anticancer agents, by free and immobilized Sorangium cellulosum was studied using the repeated batch culture process. The concentration of alginate used in immobilization was directly related to the mass transfer rate of nutrients, mechanical stability, and the epothilone B production yield. With the optimized 3% (w/v) calcium alginate carrier, a prolonged repeated batch culture was investigated for the 5 repeated batches for 24 days. The maximum productivity of epothilone B obtained from the alginate-immobilized cells was 5.03 mg/l/day, which is 3 times higher than that of free cells (1.68 mg/l/day).     

产Epothilone B纤维堆囊菌的选育与发酵优化

目的:获得高产Epothilone B菌株和最佳营养条件及发酵条件。方法:采用紫外线照射的方法对实验室保存的1株纤纤堆囊菌AHB125进行诱变处理,并采用单因素和正交实验优化培养基中的碳源、氮源和发酵条件。结果:经诱变后获得1株遗传性能稳定变异菌株(SC4-56),Epothilone B产量为14.6mg/L,比初始菌株高195%;该菌株最佳碳源和氮源为6%玉米淀粉和3%蛋白胨,产量分别为20.5和21.8 mg/L;最佳发酵条件为:转速160r/min,温度32℃,接种量8%(V/V),装液量90 mL/500mL,优化后经验证实验,Epothilone B达28.24 mg/L。结论:获得了1株高产量变异菌株,并确定了最佳生产条件。     

Complete genome sequence of the myxobacterium Sorangium cellulosum

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.     

纤维堆囊菌生长及限制因素的研究

纤维堆囊菌对可溶性淀粉、木聚糖、纤维素等复杂碳源的利用能力较强,简单碳源中只利用葡萄糖;KNO3、蛋白栋是较好的氮源,大多数氮源都能支持生长,表明该菌营养要求简单,能高效的得用处是里丰富的纤维素资源。堆囊菌的盐耐受能力较低,盐浓度高于1％的培养基中菌体几乎不能生长,Mg^2是生长必须的元素,Ca^2 对生长没有明显的作用,但是子实体的形态发生所必需的。培养基的起始pH为7.0时细胞生长较好,大于8.0或小于5.0不生长。纤维堆囊菌生长具有细胞密度依赖性,低密度的细胞不能生长。固定在滤纸上的细胞淋洗液（纤维素酶降解产物）对细胞生长具有明显促进作用,并能降低细胞生长的密度依赖性。     

Identification of an l-dopa decarboxylase gene from Sorangium cellulosum So ce90

During a screening program intended to identify genes encoding enzymes typical for secondary metabolism in Sorangium cellulosum So ce90, an aromatic amino acid decarboxylase gene (ddc) was detected. Expression of ddc in Escherichia coli and subsequent enzyme assays with cell-free extracts confirmed the proposed function derived from amino acid sequence comparisons. In contrast to other aromatic amino acid decarboxylases of eukaryotic origin, the S. cellulosum Ddc converted only L-dihydroxy phenylalanine. This is the first report of a gene encoding an L-dihydroxy phenylalanine decarboxylase in bacteria.     

Isolation of Sorangium cellulosum carrying epothilone gene clusters

Epothilone and its analogs are a potent new class of anticancer compounds produced by myxobacteria. Thus, in an effort to identify new myxobacterial strains producing epothilone and its analogs, cellulose-degrading myxobacteria were isolated from Korean soils, and 13 strains carrying epothilone biosynthetic gene homologs were screened using a polymerase chain reaction. A migration assay revealed that Sorangium cellulosum KYC3013, 3016, 3017, and 3018 all produced microtubule-stabilizing compounds, and an LCMS/ MS analysis showed that S. cellulosum KYC3013 synthesized epothilone A.     

Production of an exopolysaccharide by Antarctic yeast

Psychrophilic Antarctic yeasts produce polysaccharides in different concentrations. According to morphological, cultural, physiological and biochemical characteristics, the best producer strain was identified as Cryptococcus flavus A₅₁. The highest values for viscosity (59.1 mPa s) and crude polysaccharide productivity (5.75 g/L) were obtained in a medium containing 5 % sucrose and 0.25 % (NH₄)₂SO₄, at 24 °C for 6 d. The chemical composition and sugar constituents of the crude exopolysaccharide were determined (92.5 % saccharides, 3.34 % protein, and 4.16 % ash). The monosaccharide composition of the exopolysaccharide obtained from C. flavus strain AL₅₁ was established (55.1 % mannose, 26.1 % glucose, 9.60 % xylose, 1.90 % galactose). The microbial biopolymer has a high molar mass and homogeneity: 82 % of it had M 1.01 MDa.     

Production of an extracellular polysaccharide bioflocculant by Klebsiella pneumoniae

Klebsiella pneumoniae H12 produced a newly identified extracellular polysaccharide in an ethanol medium with a yield of 3.0 g/l. The molar composition of the polysaccharide was 56.04% galactose, 25.92% glucose, 10.92% galacturonic acid, 3.71% mannose, and 3.37% glucuronic acid. The addition of 0.5%-1.5% NaCl increased production. The polysaccharide flocculated with kaolin clay in suspension at the concentration of 1 ppm in a 300-ppm solution of CaCl2. Almost all bacterial species cells aggregated in the polysaccharide solution. The ability to flocculate with kaolin clay changed with the pH and with the concentrations of coexisting cation and anion species. The polysaccharide flocculant may participate in in vivo bacterial aggregation or adherence to host organisms.     

Selective production of epothilone B by heterologous expression of propionyl-CoA synthetase in Sorangium cellulosum

The metabolic engineering of epothilones, as secondary metabolites, was investigated using Sorangium cellulosum to achieve the selective production of epothilone B, a potent anticancer agent. Thus, the propionyl-CoA synthetase gene (prpE) from Ralstonia solanacearum was heterologously expressed in S. cellulosum to increase the production of epothilone B. Propionyl-CoA synthetase converts propionate into propionyl-CoA, a potent precursor of epothilone B. The recombinant S. cellulosum containing the prpE gene exhibited a significant increase in the resolution of epothilones B/A, with an epothilone B to A ratio of 127 to 1, which was 100 times higher than that of the wild-type cells, demonstrating its potential use for the selective production of epothilone B.     

Development of a mariner-Based Transposon for Use in Sorangium cellulosum

In order to generate marked insertions in the myxobacterium Sorangium cellulosum, a transposon based on the eukaryotic mariner transposon was developed. The transposition frequency was increased with the use of a mutated tnp gene. The transposon randomly inserts into the chromosome, as demonstrated by targeted mutagenesis of the epoK gene.     

Origins of Replication in Sorangium cellulosum and Microcystis aeruginosa.

The genome of Sorangium cellulosum has recently been completely sequenced, and it is the largest bacterial genome sequenced so far. In their report, Schneiker et al. (in Complete genome sequence of the myxobacterium Sorangium cellulosum, Nat. Biotechnol., 2007, 25, 1281-1289) concluded that 'In the absence of the GC-skew inversion typically seen at the replication origin of bacterial chromosomes, it was not possible to discern the location of oriC'. In addition, the complete genome of Microcystis aeruginosa NIES-843 has also been recently sequenced, and in this report, Kaneko et al. (in Complete genomic structure of the bloom-forming toxic cyanobacterium Microcystis aeruginosa NIES-843, DNA Res., 2007, 14, 247-256) concluded that 'there was no characteristic pattern, according to GC skew analysis'. Therefore, oriC locations of the above genomes remain unsolved. Using Ori-Finder, a recently developed computer program, in both genomes, we have identified candidate oriC regions that have almost all sequence hallmarks of bacterial oriCs, such as asymmetrical nucleotide distributions, being adjacent to the dnaN gene, and containing DnaA boxes and repeat elements.     

Development of a mariner-based transposon for use in Sorangium cellulosum

In order to generate marked insertions in the myxobacterium Sorangium cellulosum, a transposon based on the eukaryotic mariner transposon was developed. The transposition frequency was increased with the use of a mutated tnp gene. The transposon randomly inserts into the chromosome, as demonstrated by targeted mutagenesis of the epoK gene.     

Soraphinol C, a new free-radical scavenger from Sorangium cellulosum

A new compound named soraphinol C (1) was isolated from myxobacteria Sorangium cellulosum KM1001 a soil isolate, together with a structurally related known compound, 4-hydroxysattabacin (2). These compounds were isolated by silica gel column chromatography and recycling preparative HPLC, consecutively. The structures of the compounds were determined on the basis of combined spectroscopic analyses. Compounds 1 and 2 exhibited antioxidant activity as a radical scavenger in the experiment using a hydrophilic free-radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride with ORAC values of 0.956 and 0.617, respectively.     

Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum

The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44. The gene cluster contains seven genes and spans approx. 56kb. The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules. The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones. EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively. EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro.     

Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56

Sesquiterpenes are particularly interesting as flavorings and fragrances or as pharmaceuticals. Regio- or stereoselective functionalizations of terpenes are one of the main goals of synthetic organic chemistry, which are possible through radical reactions but are not selective enough to introduce the desired chiral alcohol function into those compounds. Cytochrome P450 monooxygenases are versatile biocatalysts and are capable of performing selective oxidations of organic molecules. We were able to demonstrate that CYP109D1 from Sorangium cellulosum So ce56 functions as a biocatalyst for the highly regioselective hydroxylation of norisoprenoids, α- and β-ionone, which are important aroma compounds of floral scents. The substrates α- and β-ionone were regioselectively hydroxylated to 3-hydroxy-α-ionone and 4-hydroxy-β-ionone, respectively, which was confirmed by ¹H NMR and ¹³C NMR. The results of docking α- and β-ionone into a homology model of CYP109D1 gave a rational explanation for the regio-selectivity of the hydroxylation. Kinetic studies revealed that α- and β-ionone can be hydroxylated with nearly identical V _max and K _m values. This is the first comprehensive investigation of the regioselective hydroxylation of norisoprenoids by CYP109D1.     

The Phosphatomes of the Multicellular Myxobacteria Myxococcus xanthus and Sorangium cellulosum in Comparison with Other Prokaryotic Genomes

Background

Analysis of the complete genomes from the multicellular myxobacteria Myxococcus xanthus and Sorangium cellulosum identified the highest number of eukaryotic-like protein kinases (ELKs) compared to all other genomes analyzed. High numbers of protein phosphatases (PPs) could therefore be anticipated, as reversible protein phosphorylation is a major regulation mechanism of fundamental biological processes.

Methodology

Here we report an intensive analysis of the phosphatomes of M. xanthus and S. cellulosum in which we constructed phylogenetic trees to position these sequences relative to PPs from other prokaryotic organisms.

Principal Findings

Predominant observations were: (i) M. xanthus and S. cellulosum possess predominantly Ser/Thr PPs; (ii) S. cellulosum encodes the highest number of PP2c-type phosphatases so far reported for a prokaryotic organism; (iii) in contrast to M. xanthus only S. cellulosum encodes high numbers of SpoIIE-like PPs; (iv) there is a significant lack of synteny among M. xanthus and S. cellulosum, and (v) the degree of co-organization between kinase and phosphatase genes is extremely low in these myxobacterial genomes.

Conclusions

We conclude that there has been a greater expansion of ELKs than PPs in multicellular myxobacteria.     

Transfer of mobilizable plasmids to Sorangium cellulosum and evidence for their integration into the chromosome.

Recombinant vectors derived from the broad-host-range mobilizable plasmid pSUP2021 were constructed and transferred by IncP-mediated conjugation from Escherichia coli to Sorangium cellulosum, where they were integrated into the chromosome by homologous recombination and maintained stably. This appears to be the first system of gene transfer to S. cellulosum.