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Probing Marine Systems with Ribosomal RNAs

By Stephen Giovannoni and S. Craig Cary

THE ADDITION of molecular genetic tools to cambrian. This feature has occasionally been a dis- oceanographic toolboxes began with a pronounced appointment for those interested in recent evolu- emphasis on ribosomal RNAs (rRNAs). Already tionary events. However, for the study of distantly these molecules have been used to detect and exam- related taxa, the conservative nature of rRNAs, and ine the abundance of novel bacterioplankton species, the fact that they are not laterally transferred among to study specificity and diversity among zoox- , has made them useful for tracing evolu- anthellae, to identify the chemolithotrophic sym- tionary relationships with molecular phylogenetic bionts of hydrothermal vent metazoans, and to study techniques. Indeed, different parts of rRNAs, and evolutionary relationships among a myriad of different RNAs, accumulate mutations at different species, including the most primitive metazoans. rates, making them even more versatile tools. • . . the conservative Here we review some present and projected applica- Bacterial ribosomes contain three rRNA types tions of this molecule to oceanographic science and in equimolar amounts: 23S (-2,400 nucleotides in nature of rRNAs, attempt to explain its preeminence in the emerging length), 16S (1,540 nucleotides), and 5S (120 nu- • . . has made field of molecular oceanography. cleotides). The eukaryotic counterparts of these molecules are slightly larger. The rRNA are them useful for Why Ribosomal RNAs? arrayed on in a specific order within Of the 1,500 or so genes in bacterial cells (and tracing evolutionary transcriptional units called operons (Fig. 1; Bro- many more in ) rRNA genes have been sius et al., 1981). The most conserved of the relationships with a focus of attention because of their unusual prop- genes and the most widely used is the 16S rRNA. erties. Most genes are coded information specify- molecular phyloge- Large subunit rRNAs, which are about 5-10 times ing the sequence of amino acids for proteins. more variable, are coming into wider use for ex- Ribosomal RNA genes (rDNAs) specify nonen- netic techniques. amining relationships among closely related taxa. coded, structural RNA products, which are direct Probably the most variable parts of rRNA operons copies of the . These products, the rRNAs, are are the internal transcribed spacers (ITS), which parts of a much larger structure, the ribosome. Ri- are now the focus of investigations centered on bosomes are abundant cellular components filling biogeographical questions. the essential function of protein synthesis. The The extensive use of rRNAs as molecular abundance of ribosomes is itself a persuasive argu- chronometers for phylogenetic studies led to the de- ment for their use--rRNAs may constitute 20% of velopment of large computer data bases and a re- the dry weight of an actively growing bacterial vised understanding of evolutionary relationships . Furthermore, cells regulate ribosome synthesis among , , and invertebrates. This in response to growth rate, suggesting that crucial progress has been fueled by the rapid development information about the growth status of cells might of new techniques, such as the polymerase chain re- be obtained by studying specific rRNA content. action (PCR), which brought molecular studies In the 1970s Carl Woese and colleagues began within reach of scientists not steeped in the arcane to use ribosomes for comparative studies among discipline of chemistry• Polymerase bacteria (Woese, 1987). They were an obvious chain reaction, which won Kary Mullis a Nobel choice, in part because of their abundance, but also prize in 1993, is a method for synthesizing millions because of another fortunate feature: they are very of copies of a gene. For a review of molecular biol- highly conserved, which makes them useful for ogy relevant to oceanography, see Falkowski and comparing organisms that speciated during the Pre- LaRoche (1991). The result of this tremendous re- search effort is summarized in the computer files of s. Giovannoni and S.C. Cary, Department of Microbiol- the RNA DataBase Project (RDP), which now in- ogy, Oregon State University, Corvallis, OR 97331. USA. cludes about 1,400 sequences (Olsen et al., 1991).

OCEANOGRAPHY,Vo1.6. No. 3"1993 95 5S rRNA ical reaction in which noncovalent hydrogen 16S rRNA tRNA 23S rRNA ~' bonds are formed between two strands of DNA or RNA that have complementary sequences. The de- sign of hybridization probes takes advantage of an important property of molecular biology that has 171 193 92 made it a productive science: the informational I I I I I I I quality of the data. Sequence data bases can be 0 1000 2000 3000 4000 5000 6000 scanned by any investigator to reveal short "do- Length (Nucleotides) mains"--regions 15-30 nucleotides in length-- which uniquely identify a gene of interest (Fig. 2). Fig. 1: The rrnB operon from the of Escherichia coli (Brosius It is important to note that any 20-base polynu- et al., 1981). Functional ribosomal RNA and transfer RNA genes, all of cleotide will occur on the average only once in which code .for structural RNA products, are shown in filled black. Numbers 10 ': base pairs of random sequence. Interactions indicate lengths in nucIeotides. The unfilled segments are internal tran- between base pairs different from those originally scribed spacers (ITS). which are highly variable, in contrast to the structural defined by Watson and Crick can make probes genes involved in protein synthesis, which are highly conserved. The ITS re- less specific than these numbers might suggest. An gions have no known ,fimction, other than to link rRNAs together during the example would be the pair guanine-uracil, instead early stages of rRNA synthesis. of the canonical guanine-cytosine. Nonetheless, short DNA molecules, oligonucleotides, can be made that will pair, by hydrogen bonding, with In addition to their use for molecular phyloge- very high specificity to unique target sequences. nies, rRNAs and rDNAs can be hybridized to The DNA molecules are made on automated DNA DNA probes for the quantitative detection of or- synthesis machines. Hypervariable domains of ganisms in natural samples (Stahl et al., 1988; small subunit rRNAs, in which there are only two Giovannoni et al., 1990). Hybridization is a chem- or three variations in nucleotides between bacter-

DATA:

Pr~hlorococcus UGAAUUU--CGCCUGAGGAU SAR7 UGAAUUU--CGCCUGAGGAU SAR6 UGAAUUU--CGCCUGAGGAU SARll GCUUUAUGCGCCGAAGGAU

ANALYSIS:

SYNTARGET UGAAUUUCGCCUGAGGAU IIIIIIIIIIIIIIIIII SYN PROBE ACTTAAAGCGCACTCCTA

1 y

Fig. 2: A simplified example of the procedure used to design phylogenetic DNA probes from data bases of 16S rRNA sequences. The 16S rRNA is a mosaic of conserved and variable regions. In this example, a small sequence domain is identified which is shared by autotrophic picoplankton (marine Synechococcus and Prochlorococcus), but differs from homologous regions of the SARl l gene (Britschgi and Giovan- noni, 1991). SAR7 and SAR6 are marine Synechococcus and Prochlorococcus genes, respectively, which were cloned directly from seawater. SARl l is an abundant bacterioplankton group that is known only ge- netically. The target site for the probe is indicated on the 16S rRNA structural model. Actual analysis uses computers and a duta base of >1,000 nucleotide positions from 500 organisms.

96 OCEANOGRAPHY'VoI.6, NO. 3°1993 ial subspecies, can be used for accurate but could also be a consequence of the failure of identification with DNA probes. Likewise, other some species to grow on bacteriological media. sequence domains with varying degrees of evolu- Many eukaryotic phytoplankton species have not tionary conservation can be used to design probes been cultivated because of their tricky require- of species, genus, family, phylum, kingdom, or ments for growth (Brand, 1987). This may be true universal specificity (Giovannoni et al., 1988). Ei- also for bacterioplankton. ther rRNAs, or the genes themselves, can be used Proponents of a competing theory have argued as targets for probes. that bacterioplankton are ordinary marine bacteria The remainder of this review will focus on the that have entered a viable but nonculturable state use of rRNAs as genetic markers for the as a consequence of deprivation (Rozak identification of microorganisms in marine sys- and Colwell, 1987). These physiological argu- tems. Two main applications of this approach so ments are somewhat difficult to reconcile with the far have been studies of bacterioplankton diversity wider body of ecological theory emerging from and bacterial symbionts of invertebrates. In most studies of the microbial loop, which holds that cases, more variable molecular structures, such as bacterial production is relatively high in the open mitochondrial DNA, are proving useful for popu- ocean (Azam et al., 1983). Radioactive thymidine lation genetic studies of higher organisms. uptake studies and growth estimates extrapolated from protozoan grazing have lent support to the Bacterioplankton Diversity notion that in situ division times for bacterio- Ribosomal RNAs are providing both informa- plankton are on the order of hours or days, al- tion on bacterioplankton phylogcnetic diversity though concerns about the universality of thymi- and taxon-specific nucleic acid probes. These dine-uptake mechanisms in bacteria have added an probes can be used experimentally to establish the element of uncertainty (Hollibaugh, 1988). The re- identity of cultured strains and to study the spatial markably small size of oceanic bacterioplankton and temporal distributions of microbial taxa in the has also figured in the debate, the question being oceans. A major strength of this approach is that it whether this is the normal state of bacterioplank- can identify organisms that have not been grown ton or a consequence of nutrient limitation. Stud- in culture (Olsen et al., 1986). These investiga- ies by Ferguson and co-workers suggested that the tions are testing a premise favored by some mem- addition of to seawater samples caused a bers of the scientific community: that many bacte- shift in the average size of bacterioplankton, but • . . bacterioplankton rioplankton species have not been defined in failed to establish whether this was due to shifts in taxonomic, phylogenetic, or experimental terms. are widely recognized physiology or species dominance (Ferguson et al.. Although bacterioplankton are widely recog- 1984). Lee and Fuhrman (1991) used genomic as important agents nized as important agents of biogeochemical DNA/DNA hybridization to examine changes in change in marine ecosystems, microbial communi- of biogeochemical the composition of confined bacterioplankton ties are often regarded as "black boxes." Little communities and found that no changes occured change in marine progress has been made in developing predictive within 24 hours, although the number of cultur- theories explaining planktonic processes in terms ecosystems .... able bacteria increased significantly. of the activities of specific microbial groups. In Analyses of 16S rDNAs cloned "at random" most cases, it has been difficult even to measure from oceanic bacterioplankton populations have the abundance of significant bacterioplankton added a new dimension to the debate by providing species. The principle exceptions have been pho- a less biased list of the species present in natural toautotrophic* picoplankton, which can be de- populations. Clone libraries from the National Sci- tected reliably by the autofluorescence of their ence Foundation Joint Global Oceans Flux Studies light-harvesting pigments, and some heterotrophic Program (JGOFS) ALOHA station in the central bacteria, which are easily cultivated. In question Pacific, the Sargasso Sea, and several depths in the have been the abundant cells that are not Western California current have been examined autofluorescent and do not form colonies on stan- (Giovannoni et al., 1990; Schmidt et al., 1991; dard microbiological media. Fuhrman et al., 1993). Although the cloning ap- Uncertainty about bacterioplankton identity proaches used by these workers were different, they stems from a simple observation: in the photic all relied upon analyses of genes cloned directly zone bacterial cell counts measured by epifluores- from bacterioplankton biomass without cultivation. cence microscopy vary from 0.2 × 10 ° to 2 × 10(' In each case the selection of single genes from a cells per ml, but viable cell counts on bacteriolog- complex genomic DNA mixture was accomplished ical media are typically three orders of magnitude by the ligation reaction used in gene cloning, which lower (Kogure et al., 1979)• These observations splices single pieces of DNA into vector molecules could result from many cells of a given species for subsequent isolation and replication. Gene failing to form colonies (low plating efficiencies), cloning procedures potentially can involve biases that are not fully understood, but the biases are * Photoautotroph, an able to use light as its sole much less encumbering than those imposed by mi- source of energy and CO? as its sole source of carbon. crobial cultivation requirements.

OCEANOGRAPHY°VoI. 6, No. 3°1993 97 The results have been startling. Comparisons of chaea, previously known as the Archaebacteria, the unknown genes to data bases of 16S rRNAs are a domain of prokaryotes clearly separate from from cultivated species have revealed little over- the Bacteria. lap. Furthermore, many of the novel microbial The "cyanobacterial'" sequences recovered form taxa detected so far were found in both the Pacific a cluster of high similarity, which includes the se- and Atlantic samples, suggesting that they are quences of known marine Synechococcus sp. broadly distributed• The genetic markers from However, multiple closely related but unknown these samples fall into several phylogenetic lineages were observed. This finding is consistent • . . many of the groups. A majority of the clones belong to the with the observation that immunofluorescence de- novel microbial cyanobacteria and proteobacteria, although Gram- tection with antibodies to surface antigens of culti- positive bacteria and flavobacteria were also de- vated marine Synechococcus sp. can account for taxa.., were tected. In separate work, novel (archae- only 20% of the Synechococcus cells (Campbell et found in both the bacteria) genes have been found also (DeLong, al., 1983). When the 16S rRNA sequence of a cul- 1992; Fuhrman et al., 1992). A general phyloge- tivated prochlorphyte (Prochlorococcus marinus) Pacific and Atlantic netic tree showing relationships among these was added to the data base, it was found to be very similar to one branch of unknown "'marine samples . . . groups is shown in Figure 3. The phylum Cyanobacteria includes all known cells and eu- Synechococcus" genes cloned from the open karyotic organelles capable of photolytically oxi- ocean (Urbach et al., 1992). This surprising result dizing water. The phylum Proteobacteria, which underscored the substantial phenotypic diversity includes primarily Gram-negative bacteria of the that can be found among closely related 16S o~,/3, y, and 6 subdivisions, is physiologically di- rRNA genes and further suggested that some of verse. Most common strains of marine prokary- the additional diversity within the marine Syne- otes, including the genera Photobacterium, Vibrio, chococcus gene cluster might be uncultivated Oeeanosprillum, and Alteromonas, are members prochlorophyte lines. of the y subdivision of the Proteobacteria. The Ar- The observation of abundant a proteobacterial 16S rRNA genes in the bacterioplankton commu- nity was not a complete surprise, since many members of this diverse physiological group are ~A SAR7 rochlorococcusmarinus "oligotrophic" heterotrophs--aerobic bacteria able R6 to subsist on dilute carbon sources. However, a nechococcusPCC630] Oxygenic Anabaena cylindrica Phototrophs proteobacteria had never been considered an im- GIoeobacterviolaceus portant component of bacterioplankton, and rarely have o~ proteobacteria been used as models for Agrobacterium tumefaciens - Ancylobacter aquaticus marine microbial processes, most instead being fo- Rhodobacter marina cused on marine isolates of y proteobacteria. Caulobacter crescentus eudomonas diminuta Among the many novel a proteobacterial genes SAR83 (X Proteobacteria observed, two clusters of particular abundance, the Roseobacter denitrificans rythrobaeterlongus SAR11 and SAR83 groups, emerged. The physiol- Rhodospirillum salinarum SARll ogy of the SAR11 cluster remains entirely un- known. The physiology of the SAR83 cluster also PhOtobacteriumleiognathi is unknown; however they are phylogenetically al- Photobacteriumphosphoreum brio harveyi lied to a known marine bacterium, Roseobacter __•sAR s "2 y,omo= .a ico "~ Proteobacteria denitrificans. Roseobacter is capable of photo- Alcaligenes eutrophus heterotrophic+ growth, using bacteriochlorophyll as a light-harvesting pigment. We have been un- Archaea able to detect bacteriochlorophyll a in surface wa- ~W~ MethanosarcinaGroupII barkeri Halobacterium halobium ters at the Bermuda Atlantic Time Series (BATS) Group I Archaea Archaea station, suggesting that the SAR83 cluster are not Desulfurococcus mobilus I ~ Pyrodictium occultum photoheterotrophs. Thermophilum pendens In separate work, DeLong (1992) and Fuhrman 1 et al. (1992) have found rDNA markers for a O.Ol gubstimtions per nucleotide position novel group of Archaea (archaebacteria) in coastal and oceanic midwater. Recently, we have found Fig. 3: Evolutiona~ comparisons qf picoplankton I6-S rDNAs to genes.fkom that these lineages account for a majority of 16S cultured organisms. Genes with the prefix "SAR'" were cloned directly from rDNAs in the upper mesopelagic zone at the a surface water bacterioplankton population collected at Hydrostration S in BATS station in the Sargasso Sea (N. Adair and the Sargasso Sea. Otherwise, the names refer to cultured species. The genes S.J• Giovannoni, unpublished observations). The from group I and group H Archaea shown here were cloned from coastal water samples (DeLong, 1992), but similar genes were cloned from mesopelagic water samples in the open Pacific by Fuhrman et al. (1992, ? Photoheterotroph, an organism using light as a source of 1993). This phylogenetic tree was inferred by a distance-matrix method. energy and organic material as a source of carbon.

98 OCEANOGRAPHY*VoI.6, NO. 3"1993 unknown organisms belong to the Cren- bacterioplankton diversity at the (BATS) is out- archaeota--a "kingdom" composed of mainly lined in Figure 4. The purpose of the time-series thermophilic species. Fuhrman et al. (1992) recov- study at BATS, which is supported by the Na- ered the genes from samples collected in the tional Science Foundation JGOFS, is to focus Pacific Ocean on the western side of the Califor- multiple scientific investigations on seasonal nia Current at depths of 100 and 500 m using a changes at a single oceanic site located in the general PCR cloning technique. DeLong (1992) Sargasso Sea near Bermuda. Initial molecular in- has detected similar lineages, as well as a separate vestigations of this type were limited to a few gene cluster apparently related to the meth- samples because they relied on large water vol- anogenic Archaea, in coastal samples from the umes (>100 liters) collected by tangential flow Santa Barbara Channel, the Oregon Coast, and or other specialized filtration methods. Advances Woods Hole. His approach used specific PCR in nucleic acid purification and analytical tech- primers capable of amplifying rare Archaea genes niques are permitting smaller water samples col- from a complex population. The observation of lected by standard oceanographic methods (12 Crenarchaeom lineages in cold water habitats is liters from Niskin rosettes) to be used in routine perplexing. Huber et al. (1990) reported recover- analyses. ing viable Crenarcheota from seawater in the An example of the results from this investiga- vicinity of a volcanic eruption. However, the un- tion (Fig. 5) shows the distribution of prochloro- known Crenarchaeota 16S rDNAs recovered from phytes and SAR11 16S rDNAs in the upper 250 seawater have only a moderate percentage of gua- meters of the water column during August when nine and cytosine residues. These bases help to the water column was highly stratified. The data • . . why are so few stabilize nucleic acid duplex structures and are indicate that the SAR11 cluster is most abundant gene sequences found at high percentages in the 16S rDNAs of in the upper euphotic zone, whereas at the time of extremely thermophilic species. this sampling prochlorophytes were most abundant from cultivated The above studies made no attempt to discrimi- near the deep chlorophyll maximum. nate between freely suspended bacteria and those Recently, we detected a lineage of genes related species detected in very closely to the SARll cluster. They were attached to particles• DeLong et al. (1993) natural populations? specifically addressed the problem of particle-at- found in a 16S rRNA gene library cloned from 250 tached bacteria by examining marine snow col- meters in the Sargasso Sea. These genes are most lected in the California Bight. Comparisons of abundant in the upper mesopelagic zone, suggest- rDNA libraries from marine snow and seawater ing that marine bacteria may include subspecies were dramatic; there was no overlap between the with depth-specific distributions. For the present, two populations. Moreover, the majority of the we refer to this new lineage as DeepSarl 1. particle-attached species were related to the mi- In the same 250-m clone library we also de- crobial genera Planctomyces, Cytophaga, and tected an unusual 16S rRNA gene lineage belong- Flavobacterium, which are well known for their ability to associate with surfaces. These data are colony hybridization 11 from a single set of samples. If further work sup- ports generalization from these early results with Identification of significant II phytodetrital particles, then the common view that species in culture? I bioluminescent marine enteric bacteria (Vibrio and I Fluorescence in situ Photobacterium sp.) are important in marine mi- Hybridization croaggregate formation may be called into ques- (FISH) SEAiATER tion. The above discoveries have created a rich Imaging and counting cells; i backdrop of information and hypotheses about growth state from I bacterioplankton diversity, but as yet the questions rRNA/DNA ratios I arising are more numerous than the answers• For rDNA/rRNA example, why are so few gene sequences from Hybridization T°talcNi!~i!~eids cultivated species detected in natural populations? "0" Gene PCR It is likely that more of the bacterioplankton Ecological Studies: Temporal and Spatial I species abundant in nature will be found in culture variation in bacterioplankton I "Unknown" sequences collections as methodical DNA probe screening community structure I Phylogenetic ] approaches are applied. Yet it may also prove true Comparison [~ Characterizationof | that common laboratory culture conditions are in- DNA9 Unknown Bacterioplankton | imical to the growth of some important bacterio- Probe ~v Species I plankton species. Prochlorococcus, which was rRNA Database first detected by fluorescence-activated flow cy- tometry, was only isolated into culture after a Fig. 4: Schematic outline of the strategies used for genetic investigations of difficult and prolonged effort. bacterioplankton diversi~. These approaches do not rely on cultivation, but The strategy of our genetic investigations of instead focus on natural populations of cells obtained by fiItration.

OCEANOGRAPHY'VoI.6. NO. 3"1993 99 ing to the Chloroflexus/Herpetosiphon phylum dosymbionts found in three invertebrates collected (Rapp6 et al., 1993). This phylum is one of the from deep-sea hydrothermal vents (Distel et al., early evolutionary branches of the Bacteria. No 1988). These studies demonstrated that the vent members of this group were known previously symbionts form a coherent group that falls into the from the open ocean. It is particularly interesting Y subdivision of the purple bacteria, closely that this 16S rRNA gene reaches a maximum in associated with a free-living sulfur-oxidizing bac- abundance just beneath the euphotic zone. Collec- terimn isolated from vent water (Thiomicrospira tively, these data suggest that surface, midwater, L-12). In addition, the bacterial symbionts appear and subeuphotic zone populations differ to be unique and invariant (monophyletic) within significantly in species composition. Future inves- respective host species, suggesting several inde- tigations will be aimed at understanding the phys- pendent evolutionary origins of the . ical, chemical, and biological factors which con- Phylogenetic mapping indicates that symbiont re- trol the growth of these populations. latedness reflects host relatedness and appears not to be affected by geographic location or habitat Marine Bacterial Symbiosis variation. Ribosomal RNA directed probe technology has Rowan and Powers (1992) used 16S rDNA se- proven particularly useful in the study of marine quencing and restriction fragment length polymor- symbioses. Although it has long been known that phisms (RFLPs) to examine zooxanthella diversity many highly integrated symbiotic partnerships be- • . . in 22 invertebrate hosts. Zooxanthellae are algal symbionts tween microorganisms and eukaryotic hosts exist symbionts commonly found in corals• RFLPs are in the marine environment, the specificity, diver- • . . are frequently created by using to cut DNA molecules sity, and biological function of these relationships into fragments, which form characteristic patterns difficult or impossible have often remained obscure. Whether the sym- when resolved by chromatography. Rowan and bionts reside externally (epibiont) or actually to culture free from Powers (1992) found six classes of zooxanthellae within host cells (cytobiont), they are frequently in a host-specific pattern of distribution. They also their host environment. difficult or impossible to culture free from their discovered that the algal phylogenies were not host environment. Nucleic acid probing techniques necessarily congruent with host phylogenetic rela- based on ribosomal genes offer the power and tionships, suggesting that these symbionts have re- resolution necessary to examine aspects of these tained some autonomy and may move laterally be- relationships unapproachable through classical tween hosts in rare evolutionary events. methods. In situ hybridization and 16S rDNA sequenc- The earliest application of 16S rDNA sequenc- ing have been used to characterize the prokaryotic ing in marine symbiosis sought to examine the symbionts of several bivalves and verify the au- phylogenetic relationships of the prokaryotic en- thenticity of cultured isolates. The gill symbionts of shipworms (teredinid mollusks) fix N~ and di- Specific Hybridization gest cellulose, apparently enabling their hosts to 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 utilize wood successfully as a food source. In .... I,,,,I,,,,I .... I,Ilrllll~llf,II contrast to the results obtained by Rowan and Powers (1992) with zooxanthellae, the symbionts 0 from four separate genera of shipworms were found to be identical, falling within the y-3 sub- 4O division of the Proteobacteria (Distel et al., 1991)• In situ hybridization studies using a 16S 80 rRNA targeted oligonucleotide specific for the symbiont localized the bacteria to the gill tissue Depth (m) 120 and verified that isolates originally cultured from gill tissue homogenates were the symbionts. The gill symbiont of the bivalve Thyasira flexuosa 160 l~ -D- SAR6 was reported to be the first endosymbiotic chemoautotroph in culture (Wood and Kelly, 26,, SAR11 1989)• The 16S rRNA sequence amplified from the gill and the putative isolate both fall within 250 the y subdivision of the phylum Proteobacteria. However, distance and parsimony analyses clearly grouped the gill sequence closely with Fig. 5: Ribosomal DNA pr(~les Jor BATS35 (Aug 1992). Shown here are other known lucinid gill symbionts and the isolate the distributions of two gene clusters, SARl l (0) and prochlorophytes sequence with the free-living chemoautotroph, (SAR6; i). To produce these profiles 16-S rDNAs were amphified from Thiomicrospira sp. strain L-I 2. Thus, sequence plankton genomic DNA by the polymerase chain reaction usilzg general comparisons suggest that the isolate was indeed a primers.for Bacteria, blotted onto nylon membranes, and hybridized to ra- gill surface contaminant (Distel and Wood, 1992). dioactively labeled probes. One of the most intriguing and yet elusive

100 OCF,ANOGRAPHY°Vo1. 6. No. 3°1993 . .zJ /

I .

:7

Fig. 6: In situ hybridization to identify symbiont 16S rRNA in a whole section of the vestimen- tiferan tubeworm Ridgea piscesae. The vestimentiferan symbiont probe was labeled with a nonradioactive moeity (digoxygenin-UTP) and detected immunocytochemically. The trophosome (trunk region) clearly hybridized to the probe. The Iobular nature of the trophosome, with larger bacteria concentrated toward the periphery, results in higher color development near the outside of the lobules (drop out). Reproduced from Cary et al. (1993). problems in marine symbiosis research is the can occur vertically (from parent to offspring), question of symbiont transmission. By what horizontally (spread of symbiont between contem- process are the symbionts acquired by the host porary hosts), or through a direct infection from and at what stage does the relationship actually an environmental stock of microorganisms. Mech- become biologically significant? These questions anisms of transmission have been are particularly relevant to endosymbiotic associa- successfully addressed only in certain culturable tions where the adult host relies heavily on its host species (i.e., sponges and tunicates) where the symbionts for nutriment. Symbiont transmission early stages can be obtained. Approaching

OCEANOGRAPHY'Vo1.6, No. 3"1993 101 similar questions in deep-sea organisms is more this epibiotic microflora have not been deter- problematic. mined. Currently we are using 16S rRNA se- A knowledge of symbiont transmission in hy- quence analysis to examine the diversity of this drothermal vent organisms would affect our inter- population, and we hope to derive some indication pretations of their reproduction, dispersal, and of the function of this unique assemblage. colonizing strategies. The isolated, ephemeral na- ture of the vents and the obligate nature of these Other Applications of rRNAs to Marine symbioses suggests transmission strategies that Science would not leave the inheritance to a chance event. Fluorescently labeled oligonucleotide probes for We have designed three oligonucleotide probes rRNAs are now being used widely for the detec- specific to each of the symbionts of three vent or- tion of single microbial cells by microscopy and ganisms: the vesicomyid clam Calyptogena flow cytometry (DeLong et al., 1989; Amann et magnifica, the vent mussel Bathymodiolus ther- aI., 1990a,b). Cell biologists refer to this method as mophilus, and the vent tubeworm R~ftia pachyp- in situ hybridization, referring to the fact that the tila. We have used two approaches to look for hybridization reaction actually takes place inside vertical symbiont transmission in these vent or- intact cells that have previously been preserved in ganisms. The probes are employed as PCR formaldehyde. This method can be used to identify primers for the sensitive detection of symbiont single cells and also to reveal their growth status, signal (<1 pg) from bulk gonadal DNA extracts because the fluorescence of hybridized probes is a of their respective hosts (Cary et al., 1993). The function of cellular ribosome content (Kerkoff and same primers are also used for in situ hybridiza- Ward, 1993; Kemp et al., 1993; Lee and Kemp in tion experiments to localize the position of sym- press). One of the most interesting results from this biont signal in thin sections of host tissues (Fig. work so far has been the observation by Kemp and 6). To date, these studies have shown that the co-workers that a significant proportion of bacterial eggs of the vestimentiferan tubeworms are free of cells in coastal plankton populations are inactive. symbionts before spawning and suggest that the This approach has been applied to the identifi- bacteria must be acquired at a later time, probably cation of cellulolytic bacteria in shipworms, the after settlement. This finding suggests that the identification of planktonic flagellates, and the symbiont must be free-living and possibly cultur- measurement of cellular growth rates in biofilms • . . a significant able. Most recently it has been shown that the (Distel et al., 1991: Lim et al., 1993', Poulsen et ovaries of three species of vesicomyid clams (Ca- al., 1993). Another area of active research with proportion of lyptogena spp.) all have amplifiable symbiont sig- fluorescent oligonucleotide probes for rRNAs is bacterial cells in nal (Cary and Giovannoni, 1993). Sequence com- their application to the detection of sulfate-reduc- parisons and RFLP analysis confirm that the ing bacteria. These probes in particular are promis- coastal plankton signal is identical to that of the symbiont from ing tools for the exploration of benthic microbial populations are each respective host. Tissue in situ hybridizations processes, biofilms, and anaerobic microenviron- with the symbiont specific oligonucleotides have ments (Devereaux et al., 1992). inactive. localized the symbionts to follicle cells surround- Uses of rRNAs for the identification of geneti- ing the developing oocytes, thus suggesting a ver- cally distinct phytoplankton populations are being tical mechanism of symbiont transmission in the explored. Scholin and Anderson (1993) report that vesicomyids. hypervariable regions of the large subunit rRNAs Our transmission studies are continuing to in- (28S rRNAs) of the dinoflagellate Alexandrium vestigate the ontogenetic and biochemical can be used to discriminate between closely re- processes of symbiont inheritance in the gutless lated regional populations of strains and species. protobranch bivalve, Solemya reidi, which also The data are providing evidence of the recent dis- harbors endosymbiotic bacteria in the gill tissue. persal of toxic strains from North America to We plan to advance the probing technology by Japan. Similar genetic studies, relying on the most monitoring the expression of key enzymes to as- variable regions of rRNAs, are likely to be useful sess the metabolic state of the symbionts during in resolving other plankton population biology the incorporation process and determine the role questions. This will be particularly true in cases of the symbionts in the hosts' early development. such as larval forms where morphological charac- Probably the most impressive of the vent taxa ters are not useful for identification. are the tube-dwelling alvinellid polychaetes found Internal transcribed spacers (Fig. 1) are being inhabiting the hotter areas of active hydrothermal used to address biogeographical questions that re- vent chimneys. An individual AlvineIla pompejana quire the resolution of relatively recent evolution- was recently reported to have survived tempera- ary events. Maternally inherited DNA, such as tures in excess of 100°C for a prolonged period of ITS regions, are particularly useful for this time (Chevaldonne et al., 1992). These poly- purpose. This technique has been applied to the chaetes have episymbiotic bacteria intimately as- macrophytic algae Cladophoropsis, Gracilaria, sociated with modified structures of the host body and Gracilariopsis (Goff et al., in press; Kooistra wall (Gaill et al., 1987). The diversity and role of et al., 1992).

102 OCEANOGRAPHY'VoI.6, No. 3"1993 Some Limitations of Ribosomal RNAs Brosius, J., T.J. Dull, D.D. Sleeter, and H. Noller, 1981: Gene Although molecular phylogeny may reveal in- organization and primary structure of a ribosomal RNA operon from Escherichia coli. J. Mol. Biol., 148. 107-127. sights into the physiology of organisms, it does Campbell, L., E.J. Carpenter, and V.J. lacono. 1983: Identifi- not provide direct information on the actual or po- cation and enumeration of marine chroococcoid cyano- tential biogeochemical activity of organisms. For bacteria by immunofluorescence. Appl. Environ. Micro- these purposes, markers for functional genes, such biol., 46. 553-559. as nitrogenase, nitrite reductase, and RUBP car- Cary, S.C. and S.J. Giovannoni, 1993: Transovarial inheritance of endosymbiotic bacteria in vesicomyid clams found boxylase, are more useful. In the case of bacterio- inhabiting deep-sea hydrothermal and cold vent sys- plankton known only by their rDNA markers, it tems. Proc. Natl. Acad. Sci. USA. 90, 5695-5699. will be necessary eventually either to grow the or- Cary, S.C., W.D. Warren, E, Anderson, and S.J. Giovannoni, ganisms or to develop methods for establishing 1993: Identification and localization of bacterial en- genetic linkage between functional genes and dosymbionts in hydrothermal vent taxa with symbiont- specific PCR amplification and in situ hybridization rDNA markers in mixtures of genomic DNA. Ulti- techniques. Mat. Mol. Biol, Biotech., 2. 51-62. mately, it is the distributions and activities of bac- Chevaldonne, P., D. Desbruyeres, and J.J. Childress. 1992: terioplankton that are of key interest to oceanogra- Some like it hot, some like it hotter. Nature. 359. phers. 593-594. Devereux, R., M.D. Kane, J. Winfrey, and D. Stahl, 1992: Conclusion Genus- and group-specific hybridization probes for de- It is apparent that in years ahead rRNAs will be terminative and environmental studies of sulfate-reduc- a mainstay of molecular investigations in biologi- ing bacteria. Svst. Appl. Microhiol., 15, 601-609. DeLong, E.F., G.S, Wickham, and N.R. Pace, 1989: Phyloge- cal oceanography. Their role as phylogenetic netic stains: ribosomal RNA-based probes for identifi- markers for investigating plankton populations dy- cation of single cells. Science. 243, 1360-1363. namics is just beginning to be explored. The con- DeLong, E.F,, 1992: Archaea in coastal marine environments. tinued use of rRNAs as a tool for dissecting com- Proc. Natl. Acad. Sci. USA. 89, 5685-5689. plex symbiotic associations is assured. Perhaps DeLong, E.F., D.G. Frank, and A.L. Alldridge, 1993: Phyloge- netic diversity of aggregate-attached vs. free-living ma- most important, rRNAs have been a proving rine bacteria assemblages. Limnol. Oceanogr., 38, ground for developing conceptual approaches in 924-934. molecular evolution and molecular microbial ecol- Distel, D.L. and A.P. Wood. 1992: Characterization of the gill ogy. In this regard, we may see many of the ex- symbiont of Thyasira flexuosa (Thyasiridae: Bivalvia) perimental approaches first developed with rRNAs by use of polymerase chain reaction and 16S rRNA se- quence analysis. J. Bacteriol., 174, 6317-6320. applied to a variety of macromolecules in the fu- Distel, D.L., D,L. Lane, GJ, Olsen, S.J. Giovannoni, B. Pace, ture. N. Pace, D. Stahl, and H. Felbeck, 1988: Sulfur-oxidiz- ing bacterial : analysis of phylogeny and Acknowledgements specificity by 16S ribosomal RNA sequences. J. Bacte- We are grateful to Katharine Field for advice riol., 170, 2506-2510, and suggestions and to Nanci Adair for technical Distel, D.L., E.F. DeLong, and J.B. Waterbury. 1991: Phyloge- assistance. 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104 OCEANOGRAPHY*Voh 6, NO, 3" 1993