最早的16S

Nonculturable Microorganisms in the Environment

Chapter 5 • Diversity of Uncultured Microorganisms

Although Norman Pace and colleagues (48) first suggested that rRNA sequences could be used to characterize natural communities without the need to culture, it was not until much later that the first experiments using 16S rRNA sequences tocharacterize natural communities were published (26, 69).

 

26. Giovannoni, S. J., T. B. Britschgi, C. L. Moyer, and K. G. Field. 1990. Genetic diversity inSargasso Sea bacterioplankton. Nature 345:60-63.

48. Pace, N. R., D. A. Stahl, D. J. Lane, and G. J. Olsen. 1985. The analysis of natural microbialcommunities by ribosomal RNA sequences. Micrab. Ecol. 9: I-56.

68. Ward, D. M., R. Weller, and M. M. Bateson. 1990. 16S rRNA sequences reveal numerous unculturedmicroorganisms in a natural community. Nature 345:63-65.

69. Waterbury, J. B., C. B. Calloway, and R. D. Thrner. 1983. A cellulolytic nitrogen-fixing bacteriumcultured from the gland of Deshayes in shipworms (Bivalvia: Teredinidae). Science 221:1401-1403.

 

INTERPRETING MOLECULAR DATA FOR THE ASSESSMENTOF DIVERSITY

16S 序列相似  基因组序列相似 新物种

    How does one interpret this tremendous diversity infamiliar terms? Is each new sequence a new species? The bacterial species is currentlythe only taxonomic rank in microbiology which is defined in phylogeneticterms (70). The borderline at which strains are said to belong to a (geno )speciesis 70% DNA-DNA similarity in binding assays. This admittedly subjective cutoffvalue corresponds to an esti mated 97.5 % sequence similarity between two genomes(30).Considering the total bases in the genome of E. coli to be approximately 4 X106, then 4% difference equates to about 1.6 X 105 nucleotide differences (not takinginto account the possibility that genome rearrangements are likewise a source ofdecrease in DNA similarities). This divergence could easily account for the differencesin phenotype observed among strains of some species. With the advent ofmolecular sequence analysis, it is likely that at some stage the DNA hybridizationcomponent in the species definition may be replaced by some agreed threshold 16SrONA similarity value. The reasons are obvious as more taxonomists perform sequenceanalysis than do DNA hybridization experiments, and because sequencesare more direct measurements and less prone to inaccuracies. A correlation plot ofthese two phylogenetic parameters is not linear, and for highly related organismsthe resolving power of DNA hybridization is higher than 16S rRNA sequenceanalysis (2, 61). However. it is clear that if two organisms share less than about98% l6S rRNA sequence similarity, it is unlikely that they would be assigned tothe same species by DNA-DNA binding. This correlation can help when trying toassess the molecular biodiversity revealed by environmental clones in terms ofspecies composition and numbers.

    Prior to trying to interpret what sequence divergence of environmental clonesmeans in terms of species, it is worth emphasizing that most environmental sequencesare incomplete. Indeed, some published analyses are based upon as fewas 120 nucleotides or 8% of the complete sequence. If these short stretches samplethe highly variable regions of 16S rRNA sequence, then dissimilarity will be exaggerated;if the more conserved regions are sampled, then it will be underestimated.In several studies the sequences are more than 900 bases long, and in theseanalyses the dissimilarity values are probably more reliable. Excluding the groupsof highly related clone sequences (some of which emerge because of rrn operon microheterogeneity [49]), most clones identified in several environmental studiesdemonstrate less than 980/. sequence similarity to reference sequences or to each other, and therefore most of these probably represent novel species. Some of themmay also represent new genera and a few, from their depth of branching and absenceof signatures, new phyla.

    As previously mentioned, one of the most important findings from the environmentalanalyses is the lack of absolute 16S rDNA sequence identity between acultured strain and any clone sequence, regardless of the origin of the clone library.In some cases clone sequences were identical even when they originated fromdifferent locations, such as different marine environments, and several clones fromthis habitat have been identified as being almost identical. At least in the marineenvironment, some bacterial groups are ubiquitous and highly successful. As therelevant organisms are not in culture it is impossible to say if these small differencesin rRNA sequence are reflected by phenotypic differences.

30. Johnson, J. J. 1973. The use of nucleic acid homologies in the taxonomy of anaerobic bacteria.Int. f. Syst. Bacteriol. 23:308-315.