David J. Lane

The Analysis of Natural Microbial Populations by Ribosomal RNA Sequences

Recombinant DNA methodology and rapid nucleotide sequence determinations have changed the face of cell biology in the past few years. This technology offers powerful new tools to the microbial ecologist as well. In this chapter we describe technical strategies we are developing which use these methods to analyze phylogenetic and quantitative aspects of mixed, naturally occurring microbial populations.

Analysis of hydrothermal vent-associated symbionts by ribosomal RNA sequences

Ribosomal RNA (rRNA) sequences were used to establish the phylogenetic affiliations of symbioses in which prokaryotes appear to confer sulfur-based chemoautotrophy on their invertebrate hosts. Two submarine hydrothermal vent animals, the vestimentiferan tube worm Riftia pachyptila and the clam Calyptogena magnifica, and a tidal-flat bivalve, Solemya velum, were inspected. 5S rRNAs were extracted from symbiont-bearing tissues, separated into unique forms, and their nucleotide sequences determined and related to other 5S rRNAs in a phylogenetic tree analysis. The prokaryotic symbionts are related to one another and affiliated with the same narrow phylogenetic grouping as Escherichia coli and Pseudomonas aeruginosa. The sequence comparisons suggest that Riftia is more closely related to the bivalves than their current taxonomic status would suggest.

The excision of intervening sequences from salmonella 23S ribosomal RNA

Novel, approximately 90 bp intervening sequences (IVs) were discovered within the 23S rRNA genes of S. typhimurium and S. arizonae. These non-rRNA sequences are transcribed and then excised during rRNA maturation. The rRNA fragments that result from the excision of the extra sequences are not religated. This results in fragmented 23S rRNAs. The excision of one IVS was shown to be catalyzed in vivo and in vitro by ribonuclease III. These IVSs are highly volatile evolutionarily, sometimes occurring in only some of the multiple rRNA operons of a particular cell. The sporadic nature of the occurrence of fragmented rRNAs among closely related organisms argues that such fragmentation is a derived state, not a primitive one. Possible sources of these IVSs, their parallels with internal transcribed spacers and introns in eukaryotes, and their possible roles in the evolutionary process are discussed.

[12] Reverse transcriptase sequencing of ribosomal RNA for phylogenetic analysis

Publisher Summary This chapter focuses on reverse transcriptase sequencing of ribosomal RNA for phylogenetic analysis. The cyanobacteria comprise a large group of structurally complex and ecologically important gram-negative prokaryotes whose taxonomic treatment has generated much interest and controversy over the years. By molecular taxonomic criteria (particularly 16 S rRNA oligonucleotide catalog analysis), the cyanobacteria, together with chloroplasts and prochlorophytes, are a coherent, eubacterial grouping that warrants phylum status. The sequencing protocol is a variation of the base-specific, dideoxynucleotide-terminated, chain-elongation method. It has been modified for the use of reverse transcriptase and ribosomal RNA templates. The method relies on the fact that certain regions of 16 S rRNA nucleotide sequence vary little or not at all among different organisms. A short oligodeoxynucleotide, complementary to such a universally conserved site, specifically anneals to its target site on the 16 S rRNA, even in the presence of other cellular RNAs. The RNA purification method is designed for use on pure cultures containing only one type of 16 S rRNA. Mixed cultures, however, can often be sufficiently purified by differential centrifugation or filtration, at the time of harvesting, for the method to work.

A microbiological study of Guaymas Basin high temperature hydrothermal vents

Abstract Water samples and suspended particulate matter were collected from three high temperature (156–319°C) hydrothermal vents in the Guaymas Basin during July 1985, and were analysed for a variety of chemical constituents and for the presence of viable microorganisms. Our results indicate that black smoker fluids (> 150°C) are devoid of recognizable bacteria and contain negligible concentrations of ATP ( -1 ) and low concentrations of particulate organic matter. In contrast, vent water samples collected in the hydrothermal plume at a distance of only 25 cm from the point of hot fluid discharge contain a diverse, metabolically active bacterial assemblage, high ATP concentrations (up to 372 ng 1 -1 ) and high concentrations of particulate organic carbon and nitrogen (up to 999 and 163 μg 1 -1 , respectively). The hottest waters (> 150°C) displayed a low, but measurable level of metabolic activity (incorporation of 3 H-adenine and 14 C-glutamate) at temperatures ranging from 25 to 80°C, 1 atm with maximal activity at 45°C. The maximum rate of incorporation, however, was only ∼ 1% of the activity measured in the samples collected in the hydrothermal plume. The strong metabolic preference for mesophilic growth temperatures (45°C) argues against a high temperature origin. Thermophilic sulfur-respiring bacteria were isolated from a variety of source materials including black smoker vent waters (112–319°C). All positive enrichments grew at 80°C, but none survived at temperatures in excess of 93°C. We conclude that these bacterial cells did not originate from the high temperature hydrothermal vent waters. Two different sample devices (reffered to as vent cap and smoker poker) were designed and employed during in situ collection-incubation experiments. Neither the vent cap attachment-colonization experiment nor the smoker poker collection device revealed the existence of bacterial cells in the black smoker fluids, despite evidence for a significant bacterial population in the hydrothermal plume. Our field and laboratory results indicate that thermophilic and mesophilic bacteria observed in hydrothermal vent plumes are not derived from the hot hydrothermal fluids but must originate in peripheral habitats.