Phylogenetic Diversity Of Cultured And Uncultured Myxobacteria In Soil And Marine Sediments

Myxobacteria are Gram-negative gliding bacteria.They are unique among prokaryotes for their complicated multicellular behavior,especially the morphogenesis of fruiting bodies,and therefore are considered as social bacteria. When myxobacteria faced with starvation or suffered under unfavorable environmental conditions,cells form a macroscopic fruiting body containing thousands of resting myxospores.Those myxospores are sufficiently resistant to desiccation,heat,and UV light.As a result of their specialist lifestyle,myxobacteria are found nearly everywhere.They prefer to grow in the soil,the dung of herbivores, in bark and in rotting wood.Myxobacteria are rarely mentioned in articles of ecological study on soil microbiology.The reason is that the unusual characteristics of myxobacteria make the isolation and purification of myxobacteria more difficult than other soil microorganisms,furthermore many myxobacteria strains may be omitted using traditional isolation methods because of the instability of the fruiting body structure.Therefore the cultured myxobacteria strains may be only represent a small part of myxobacteria community in environment.The advent to so called 'cultivation independent' methods has provided researchers with the ability to determine the composition of microbial communities and identify numerically important,but not yet cultured organisms.Furthermore with largely and quickly increase of the 16S rRNA gene sequences of myxobacteria which have been submitted into GenBank database library,it is feasible to study myxobacterial ecology using molecular methods based on 16S rRNA gene sequences.In this paper,we used traditional morphological methods and molecular ecological methods to study the phylogenetic relationship and ecological diversity of myxobacteria in soil and marine sediments.1.We selected 37 myxobacteria strains from our myxobacteria strains library,and those strains were classified based on their morphological characteristics.The 16S rRNA and HSP60 gene sequences of some myxobacteria strains were amplified by PCR methods,and phylogenetically analyzed.The classification of myxobaccteria mainly based on the morphological characteristics,especially the fruiting bodies.However,the fruiting body of myxobacteria is not a stable characteristic which is easy to degenerated or even lost.For example the five Cystobacterineae strains 0082-2,0085-4,0121-3,Myx9736 and NM03,the three Sorangineae strains So0007-16,So0157-52 and So139-5 were degenerated of fruiting body structures in different extents.The phylogenetic analysis of 16S rRNA gene sequences may help to define taxonomical status of difficult to identify myxobacteria.After phylogenetic analysis of 16S rRNA gene sequences of 15 Cystobacterineae strains and 22 Sorangineae strains,the results suggested the present morphology-based classification of myxobacteria was highly consistent with the phylogenetic results of 16S rRNA gene sequences at the levels of genera or higher taxa;while after phylogenetic analysis of HSP60 gene sequences of 5 Cystobacterineae strains and 22 Sorangineae strains,the results suggested HSP60 gene sequences provided a more efficient method for identification of closely related myxobacteria species.Here we also found there were two copy of HSP60 genes in Sorangium,both two HSP60 gene sequences can be used as molecular marker for phylogenetic analysis of closely related myxobacteria species.The sequences homology within same strain between different HSP60 genes was highly consistent at about 78%homology,which probably indicates that both genes are important,but play different roles in the Sorangium cells.2.Using the above amplified 37 16S rRNA gene sequences and seventy 16S rRNA gene sequences of cultured myxobacteria which were extracted from the GenBank to develop oligonucleotide primers/probes specific for the two suborders of myxobacteria,Cystobacterineae and Sorangineae,respectively,we constructed two different 16S rRNA gene clone library based on W1/1492R and W4/1492R primer sets from total soil DNA.After screening clone library with specific probes,Hybridization-positive clones were randomly selected for sequencing.From the soil sample in this study,we found seven species of myxobacteria on enriching plates.But analysis of the soil 16S rRNA gene sequences provided quite different data.There are potentially much more types of myxobacteria in nature.The myxobacteria appearing on enriching plates only represented a small part of the natural myxobacteria panorama.In the suborder Cystobacterineae,all the strains based on phylogenetic analysis of their 16S rRNA gene sequences can be divided into only four groups.However,our analysis from one soil niche revealed that there were at least 12 groups,including the above four known groups.In the suborder Sorangineae,phylogenetic analysis of all myxobacteria related sequences revealed that there were at least 5 groups, all the cultured strains can be located into only one group,and the other 4 groups were composed of all uncultured myxobacteria sequences.3.Phylogenetic analysis the community of myxobacteria in a soil niche based on total soil DNA methods suggested that myxobacteria in nature are much more diverse than were ever known,even in one soil niche.However the disadvantage of DNA-based molecular methods is the lack of differentiation between active and inactive(dead or dormant)bacterial cells,and RNA-based community analysis is more suitable to describe the metabolically active members of a population because of its rapid turnover and low RNA content in dormant cells. Therefore,we constructed two different 16S crDNA clone library based on W1/1492R and W4/1492R primer sets from total soil RNA revertased product. After screening clone library with specific probes,83 Hybridization-positive clones were randomly selected for sequencing to analysis of the active myxobacteria community.Comprehensive analysis of 16S rRNA gene sequences of cultured,uncultured and active uncultured myxobacteria indicated that there were a lot of numbers of active myxobacteria in nature,while we can only isolated 7 myxobacteria strains using traditional methods.Furthermore the results based on phylogenetic analysis of 16S crDNA sequences were quite different from 16S rRNA gene sequences,it is suggested that it was more different in diversity of between composition community of myxobacteria and metabolically active community of myxobacteria in soil environment.4.The myxobacteria are considered to be the typical soil microorganisms,however in recent years,some halophilic and halotolerant myxobacteria strains also have been found.In this paper,we isolated myxobacteria strain from 10 different depths marine sediments using traditional myxobacteria isolation methods, however there was no cultured myxobacteria strain found.This suggested there was little number of fruiting myxobacteria in marine sediment environments.For comprehensive analysis of bacteria community of marine sediment 916-1 and 7K367 M2-1,we constructed two universal bacteria 16S rRNA gene clone library. After randomly sequencing 88 and 93 clones of marine sediment 916-1 and 7K367 M2-1,we found there was no myxobacteria related sequences in marine sediment 7K367 M2-1,this may suggested myxobacteria are not a dominant group in microbial community of sediment 7K367 M2-1;and also found 3 myxobacteria related sequences in marine sediment 916-1,counted up to 3.4%of total bacteria clones,high than those in a soil niche.After construction of two types of 16S rRNA clone library with W1/1492R and W4/1492R primer sets from 5 different marine sediments,we used specific probes to screen the myxobacteria related clones.Unfortunately,there was no hybridization positive clone found.The reason maybe is too narrow specificity of probes,because the probe W2 and W5 were designed based on the 16S rRNA gene sequences of territorial cultured myxobacteria,and those marine myxobacteria may have high sequences discrepancy compared with territorial myxobacteria.So we randomly selected clones to sequence,and obtained 68 myxobacteria related sequences.After phylogenetic analysis of those marine myxobacteria related sequences and all cultured myxobacteria sequences,we found there was no marine sequence in cultured Cystobacterineae Group.This may suggested suborder Cystobacterineae strains may more suitable to survive in territorial environment,this result also consisted with the experiments since most of the Cystobacterineae strains were isolated from territorial environments.In suborder Sorangineae,there was only one clone D030-W4-42 belongs to this suborder;however have low sequence homology of 91%with the other members of this suborder.Some uncultured marine myxobacteria sequences and marine myxobacteria strains SMP-2 belonged to the same group and some groups only were composed of all uncultured marine myxobacteria sequences.The group which the genus Nannocysits belong to had no uncultured marine myxobacteria sequence.5.Furthermore we analyzed the phylogenetic relationship of uncultured marine myxobacteria sequences,uncultured territorial myxobacteria sequences and all cultured myxobacteria sequences.The result showed myxobacteria can be divided into 4 major phylogenetic clades which represented Cystobacterineae, Sorangineae,Nannocystineae and a new unknown suborder.All cultured myxobacteria were located into three known suborders Cystobacterineae, Sorangineae and Nannocystineae,but they only represented a very small part of total myxobacteria panorama.The new unknown suborder was composed of uncultured marine myxobacteria sequences.In suborder Nannocystineae,there were a lot of numbers of uncultured marine myxobacteria sequences besides cultured genus Nannocystis and marine myxobacteria strains.In this study,we established series of molecular methods for analysis of myxobacteria systemic classification and ecological diversity.We used molecular ecological methods to study ecological distribution and phylogenetic diversity of myxobacteria, and found there were potentially much more new types of myxobacteria in nature. Meanwhile,we used molecular methods to comprehensive analysis of myxobacteria community in marine sediments which have not been published before,and the results showed great existing information of myxobacteria ecology and evolution.However we are still wondering that what characteristic of those uncultured myxobacteria have? How to live so many uncultured myxobacteria in nature?...