Isolation, Purification And Classification Of Cellulolytic Myxobacteria; And The Degradation Mechanisms On Cellulose In The Microorganisms

Myxobacteria are a special bacterial group that possesses complicted multicellular behavior, in such as cellular growth, feeding, movement, differenciation and development, which makes myxobacteria to be considered as the most advanced bacteria in prokaryotes and as a well-known model in many studies. On the other hand, myxobacteria are also able to produce many novel diverse bioactive secondary metabolites and are, therefore, a greatly potential drug-screening source.Based on morphological characteristics, myxobacteria could be classified into two suboders, four families, twelve genera, and about thirty-five species. Considering their specializations in degradation of biomacromolecules, myxobacteria, except the unculturable genus Haploangium, are divided into two groups. One is bacteriolytic, efficiently lysing whole living cells of other microorganisms, and the other is cellulolytic, decomposing cellulose instead of living cells. Out of all the classified myxobacterial species, only one species or genus, depending on different classification systems, is included in the latter. Till now, work on the cellulolytic myxobacteria is focused on the screening of bioactive compounds and recently more on the biosynthetic genes for some polyketide metabolites. Nearly no reports were published related to the basic studies on the cellulolytic myxobacteria. To further research and study the cellulolytic myxobacterial source, this thesis was designed on its basic aspects, i.e. ecologic distribution, isolation, classification of the cellulolytic myxobacteria, and degradation mechanism on cellulose.1. Distribution and isolation of cellulolytic myxobacteriaWe assayed distribution of and isolated cellulolytic myxobacteria of several environmental types of samples, i.e. 13 of farmland soil, 51 of rotted bark or grasses, and several extreme samples containing alkaline soil from Chenghai Lake, mud from the bottom of ocean, Antarctic soil, and mud or sand from Chinese coasts. Enriching isolation was performed on cellulose-CNST and rabbit dung-WCX agar. Simultaneously, of the deep sea mud and Antarctic soil, additional plates were incubated at 10℃; of alkaline soil samples, additional pH10.0 plates were employed;of coast and deep sea samples, additional 50% seawater plates were used. Furmer purification from the enriched myxobacteria employed dierect purification method, heat-treatment method, antibiotics-repressing method, or antibiotics-inducing killing method. The morphological classification was performed according to the criterions in Bergey's Manual of Determinative Bacteriology (9th ed., 1989) and The Prokaryotes (2nd ed., 1992). The used morphologies included fruiting body structure, swarm, vegetative cells, myxospores, and also ability of cellulose-degradation.Thirty-one cellulolytic myxobacterial and interrogative strains were isolated from the thirteen farmland soil samples, i.e. about 2.4 strains of each sample on average. Sixty-eight strains from fifty-one bark or grasses, i.e. 1.3 strains of each sample; the appearance rate changed according to the sampling places and the kinds of plant. Of the alkaline samples, only four cellulolytic myxobacterial strains appearred (thirteen strains from four samples; no from other seven samples), of the strains, there included alkali-resistant, no alkali-resistant, and alkalophilic. Only two cellulolytic myxobacterial strains were isolated from ten ocean samples. No cellulolytic myxobacteria were found from the deep sea mud or Antarctic soil.2. The systematic classification of cellulolytic myxobacteriaTen cellulolytic myxobacterial strains were selected based on the diversity of morphological characteristics. The strains were assayed and homologically analysed of their 16S rDNA sequences. The correlation of sequence homogeneity and morphology for classification was discussed.The size of vegetative cells was the average of 200 cells in 3-day cultures, measured under a phase-contrast microscope. Fruiting bodies were ovserved using dissection microscope. The mature myxospores were extruded from the aged fruiting bodies for observation. The primer pair for 16S rDNA PCR amplification is 5'-AGA GTT TGA TCC TGG CTC AG-3' (10-30F) and 5'-AGA AAG GAG GTG ATC CAG CC-3' (1500R). The three sequencing primers were 5'-GTA TTA CCG CGG CTG CTG-31 (536-519), 5'-AAA CTC AAA GGA ATT GAC GG-3' (1115-1100) and 5'-AGG GTT GCG CTC GTT GCG-31 (907-926).All the cellulolytic myxobacterial strains were nearly the same in shape of vegetative cells or myxospores, i.e. cylindrical, rigid rods, and with bluntly round ends. The sizes of myxospores could be divided into two groups: 3-4x1 -1.2um and2-3x1 urn. Normally, the myxospores were embedded in sporangioles, however, strain So9987-5 had no any sporangiole structure. Another exception was strain So9881-1, which quickly lost the capacity of forming sporangioles after its isolation from soil. The sporangioles of cellulolytic myxobacteria were spherical or polyhedral. The polyhedral sporangioles were normally large, 30-60um in diameter and the spherical ones were small, 10-30fim in diameter. Additionally, the structure of the large polyhedral sporangioles was usually loosely organized with many inter-cystic substrates. The small spherical sporangioles were tight. Many sporangioles packed together to form fruiting bodies, small or large, dense or loose parcels. As an exception, sporangioles of So9721-1 were scattered and did not form parcels. The colors of fruiting bodies varied among yellow, orange, orange-brown, red-brown, deep brown, and black, which had no relationships with the shape, size or organization of sporangioles. Besides the morphological differences, the distributions of fruiting bodies in the swarm also varied among the cellulolytic strains.The following are the length and the accession numbers of 16S rDNA segments of the refered strains in the GenBank nucleotide sequence database: So9882-1 (AF467673, 1555bp), So9735-22 (AF467675, 1557bp), So9733-1 (AY039304, 1556bp), So0089-1 (AY079453, 1552bp), So9721-1 (AY032880, 1537bp), So9857 (AF467674, 1555bp), So9881 (AF467672, 1555bp), So9987-5 (AF387628, 1576bp), So ce26 (AF387629,1554bp) and So9741 (AF387627,1559bp).The phylogenetic tree is constructed by the Dnadist/Neighbor programs in the PHYLIP package (v3.6). The evolutionary distances of 16S rDNA sequences of the cellulolytic and the other myxobacteria were compared. The results indicated that the cellulolytic myxobacteria are homogenous in 16S rDNA sequences. The farthest evolutionary distance is less than 3%, which was obtained from the comparison of So9741 with other strains. However, the fine distances between the ten cellulolytic myxobacterial strains revealed that there are three subgroups: So9741 and So ce26 comprise two of the three subgroups, while the other tested strains compose a third subgroup. The distances of the 16S rDNA sequences of the cellulolytic myxobacteria compared the bacteriolytic myxobacteria were 4-8% to the closely related Chondromyces spp. and Polyangium thaxeri, but 15-18% to Polyangium vitellinum, Nannocystis exedens, and the Myxococcus subline.All the cellulolytic strains are united in a single genus Sorangium on the basis of their 16S rDNA homogeneities. They are not included in Polyangium, which is significantly heterogenous. The Chondromyces subline still exists and consists of Sorangium spp., Chondromyces spp. and some species in Polyangium. In Prokaryotes(1992), Reichenbach and Dworkin suggested that there might be at least two species in genus Sorangium: S. compositum produces yellow-orange fruiting bodies and S. cellulosum brown, grey, or black ones. In this thesis, it turns out that the size of myxospores and the shape of sporangioles are consistent with changes of 16S rDNA sequences, but the color of fruiting bodies and the morphologies of swarms are not. By contrasting morphological differences and 16S rDNA differences of cellulolytic myxobacteria, the present work also suggests that more than one species are included in the genus. One includes the strains with small sizes of myxospores (2-3xlum) and spherical sporangioles (10-30um in diameter). The colors of fruiting bodies are normally orange or brown, though sometimes yellow or black. The characteristics of this first species are very similar to S. compositum. The second species has polyhedral sporangioles and some larger myxospores (3-4x1 um). The sporangioles are large (40-80um in diameter) and have many inter-cystic substrates. The colors of the fruiting bodies are normally deep brown to black. This species is closely related to S. cellulosum. However, considering the differences of their 16S rDNA, this group might still be heterogeneous and include several species.3. The cellulose-degrading enzymes in cellulolytic myxobacteriaScanning electron microscopy was employed to detect the cellular behaviors of cellulolytic myxobacteria on cellulose fibers. The activities of cellulases and xylanase were assayed by DNS method. The protein concentration was determined using a Pierce kit (Pierce Co). The cell/cellulose mixture, collected from cellulose-CNST culture, was pre-washed with 20 mmol I"1 Tris-HCl buffer (pH 7.5) and then extracted with redistilled water. The water extract was combined, centrifuged, ultrafiltered, lyophilized to obtain water extract, and stored at -70 °C. After the two-step extraction, nearly no cellulase activities were detectable in the cell/cellulose mixture. The enzyme solution was eluted through Sepharose CL 4B (80 cmxl.6 cm). The fractions with 280 nm absorbance were collected respectively, assayed of their protein concentration and enzymatic activities against carboxyl methylcellulose and xylan. The molecular weight markers (29,000-2,000,000 Dalton, Sigma Co.) were run underthe same condition to determine the molecular weight range of the proteins. After Sepharose CL 4B, the collected fraction of high molecular weight was further assayed with heat-treated sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC through a DIOL-300 column (Shimadzu Co.).Many intercellular fibrils and cell-bound wart-like protuberances of about 0.1-0.2 urn in diameter were detectable on the growing cell surfaces with scanning electron microscopy. T he accessories m ight 1 ost from t he c ells w hile t he cells were gliding along the cellulose fibber. The cells tightly dumped over the cellulose fibres, degrading cellulose, and piled-up proliferating in situ. Only the cellulose touched by the cells was degraded, while the fibres nearby but not touched remained intact. This phenomenon might be explained by the hypothesis that some metabolites had accumulated in situ during the growth and hampered the further cell movement. After the growth, the accessories disappeared from the cell surface.The cellulolytic activity of different strains is coherent with their degrading ability on f ilter p aper. S orangial c ellulase a ctivities a re linked t o t he c ellular s urfaces a nd undetectable in the agar during the growth stage. Among the cellulases, endo-glucanase and xylanase appeared early and then p-glucosidase. Exo-glucanase activity was quite low and nearly undetectable in the preparations.The two-step method can efficiently extract the cellulses from the cells/cellulose mixture. The pre-wash with low concentration Tris-HCl buffer (pH7.5) will remove most of the non-cellulolytic proteins, together with 20-40% cellulase activities. The second wash with distilled water harvested 60-80% cellulolytic enzymes. Further elution on Sepherose CL 4B, the redistilled water extract from cultured filter paper powder showed several large molecule peaks, which had both endo-glucanase and xylase activities. The proteins are in the range of l,000-2,000kDa. The part of 600kDa-20kDa also contained cellulolytic activities but were small. The elution part of 20kDa contained pigments and no cellulolytic activities.SDS-PAGE electrophoresis showed that the high molecular weight fraction from Sepharose CL 4B (fraction 1) could not run into the gel well without boiling. However, there were several weak low-molecular weight bands in the gel, which might be released from the high molecular weight complex due to its unstable and the effect of sample buffers. If the sample was heat-treated before electrophoresis, the...