Content uploaded by Stephen Craig Cary
Author content
All content in this area was uploaded by Stephen Craig Cary
Content may be subject to copyright.
A preview of the PDF is not available
Transovarial Inheritance of Endosymbiotic Bacteria in Clams Inhabiting Deep-Sea Hydrothermal Vents and Cold Seeps
Abstract and Figures
Vesicomyid clams are conspicuous fauna at many deep-sea hydrothermal-vent and cold-seep habitats. All species examined have specialized gill tissue harboring endosymbiotic bacteria, which are thought to provide the hosts' sole nutritional support. In these species mechanisms of symbiont inheritance are likely to be key elements of dispersal strategies. These mechanisms have remained unresolved because the early life stages are not available for developmental studies. A specific 16S rRNA-directed oligodeoxynucleotide probe (CG1255R) for the vesocomyid endosymbionts was used in a combination of sensitive hybridization techniques to detect and localize the endosymbionts in host germ tissues. Symbiont-specific polymerase chain reaction amplifications, comparative gene sequencing, and restriction fragment length polymorphisms were used to detect and confirm the presence of symbiont target in tissue nucleic acid extracts. Nonradioactive in situ hybridizations were used to resolve the position of the bacterial endosymbionts in host cells. Symbiont 16S rRNA genes were consistently amplified from the ovarial tissue of three species of vesicomyid clams: Calyptogena magnifica, C. phaseoliformis, and C. pacifica. The nucleotide sequences of the genes amplified from ovaries were identical to those from the respective host symbionts. In situ hybridizations to CG1255R labeled with digoxigenin-11-dUTP were performed on ovarial tissue from each of the vesicomyid clams. Detection of hybrids localized the symbionts to follicle cells surrounding the primary oocytes. These results suggest that vesicomyid clams assure successful, host-specific inoculation of all progeny by using a transovarial mechanism of symbiont transmission.
Figures - uploaded by Stephen Craig Cary
Author content
All figure content in this area was uploaded by Stephen Craig Cary
Content may be subject to copyright.
... Members of the microbiome passed from parents to offspring are essential for the function and early development of their host (Carrier et al., 2021;Mcfall-Ngai et al., 2013). The maternal transfer of microbes is well studied in humans and other mammals with post-birth care, however, many marine invertebrates are broadcast spawners with no post-birth care from parents, yet they too rely on their microbiome acquired through the egg for survival (Usher et al., 2005;Cary and Giovannoni, 1993). Broadcast spawning exposes embryos to the environment where they are vulnerable to ocean warming and acidification from climate change (Hoegh-Guldberg et al., 2018;Scanes et al., 2020;Parker et al., 2013). ...
... Molluscs such as oysters have diverse microbiomes with resident microbes within their haemolymph and other tissues playing a vital role in host function (King et al., 2019a;Mcfall-Ngai et al., 2013;Scanes et al., 2021aScanes et al., , 2021bUnzueta-Martínez et al., 2021). Endosymbiotic bacteria are likely passed from mothers to offspring in clams (Cary and Giovannoni, 1993), and oyster mothers have also recently been shown to play a larger role than fathers, passing microbes to their offspring through the eggs (Unzueta-Martínez et al., 2022a). These microbes then remain throughout larval development, and are suspected to protect from pathogens, produce vitamin B12 and aid in extracellular signalling (Unzueta-Martínez et al., 2022a). ...
... In addition, 152 common ASVs were co-owned by all stages, from the eggs to the zoea, and a core microbiota of 109 ASV highlights greatly that specific ASVs were retained and transmitted through vertical transmission during the whole larval cycle (Figures 5A and S1). Vertical transmission of specific bacterial taxa from parents to their offspring has also been demonstrated in Rimicaris exoculta, a shrimp species living in hydrothermal vent areas [50,51], as well as in other marine hydrothermal or cold seep organisms such as clams [52] and aquatic macroorganisms such as fish or sponges [53,54]. Among the core microbiota, several lineages such as Alteromonas, Ascidiaceihabitans (formerly Roseobacter), Halomonas, Litoricola, Leisingera, Micrococcus, Pseudoaltermonas, Rhodovulum, Ruegeria and Sulfitobacter might exhibit probiotic or beneficial activities [55][56][57][58][59][60][61][62][63][64]. ...
During their entire lifecycle, mariculture animals are farmed in water that contains various microorganisms with which they are in close associations. Microbial exchanges between the animals and their surrounding water can occur. However, little is known about the interactions between shrimp larvae and water, and more especially, about larval bacterial selection and microbiota modulation across ontogeny. To address this gap, using HiSeq sequencing targeting the V4 region of the 16S rRNA molecule, we investigated the active prokaryotic diversity and structure of healthy Penaeus stylirostris larvae and seawater. Comparisons between different larval stages revealed evidence of stage-specific microbiotas and biomarkers, a core microbiota common to all stages, and shared taxa between successive stages, suggesting vertical transmission of bacterial taxa. Comparisons between stage-specific microbiotas and core microbiotas with water storages highlighted that many taxa associated with the larvae were originally present in the natural seawater, underlining horizontal transmission of bacteria from water to larvae. As some of these lineages became active at specific larval stages, we suggest that larvae were able to modulate their microbiota. This study provides insight into larvae-microbiota interactions at the larval stage scale.
... Vertical transmission of bacterial symbionts implies the presence of symbionts in the gonads and/or gametes, allowing transfer into embryos and larvae as well, as previously described in the insect Blattela germanica [48] or the bivalve Solemya reidi [49,50]. In Aratus pisonii and Minuca rapax, symbionts were detectable neither in the gonads nor gametes and larvae using PCR with three distinct primer sets and SEM on the eggs of A. pisonii. ...
Aratus pisonii and Minuca rapax are two brachyuran crabs living with bacterial ectosymbi-onts located on gill lamellae. One previous study has shown that several rod-shaped bacterial mor-photypes are present and the community is dominated by Alphaproteobacteria and Bacteroidota. This study aims to identify the mode of transmission of the symbionts to the new host generations and to identify the bacterial community colonizing the gills of juveniles. We tested for the presence of bacteria using PCR with universal primers targeting the 16S rRNA encoding gene from gonads, eggs, and different larval stages either obtained in laboratory conditions or from the field. The presence of bacteria on juvenile gills was also characterized by scanning electron microscopy, and subsequently identified by metabarcoding analysis. Gonads, eggs, and larvae were negative to PCR tests, suggesting that bacteria are not present at these stages in significant densities. On the other hand, juveniles of both species display three rod-shaped bacterial morphotypes on gill lamellae, and sequencing revealed that the community is dominated by Bacteroidota and Alphaproteobacte-ria on A. pisonii juveniles, and by Alphaprotobacteria, Bacteroidota, and Acidimicrobia on M. rapax juveniles. Despite the fact that juveniles of both species co-occur in the same biotope, no shared bacterial phylotype was identified. However, some of the most abundant bacteria present in adults are also present in juveniles of the same species, suggesting that juvenile-associated communities resemble those of adults. Because some of these bacteria were also found in crab burrow water, we hypothesize that the bacterial community is established gradually during the life of the crab starting from the megalopa stage and involves epibiosis-competent bacteria that occur in the environment.
... Most major metazoan lineages have been observed to transmit microbes from mother to offspring [annelids (Davidson & Stahl, 2008), arthropods (Ferree et al., 2005), chordates (Hirose, 2015), cnidarians (Apprill et al., 2009), echinoderms (Carrier et al., 2021), flatworms (Jäckle et al., 2019), molluscs (Cary & Giovannoni, 1993), nematodes (Stevens et al., 2001), and sponges Díez-Vives et al., 2022)]. These developmental symbionts have deep evolutionary origins and play integral roles in host biology and ecology, including acclimation (Foutaine et al., 2022), gametogenesis (Dedeine et al., 2001), and metamorphosis (Song et al., 2021). ...
The transmission of microbes from mother to offspring is an ancient, advantageous, and widespread feature of metazoan life-history. Despite this, little is known about the quantitative strategies taken to maintain symbioses across generations. The quantity of maternal microbes that is provided to each offspring through vertical transmission could theoretically be stochastic (no trend), consistent (an optimal range is allocated), or provisioned (a trade-off with fecundity). Examples currently come from animals that release free-living eggs (oviparous) and suggest that offspring are provided a consistent quantity of symbionts. The quantity of maternal microbes that is vertically transmitted in other major reproductive strategies have yet to be assessed. We used the brooding (viviparous) sponge Halichondria panicea to test whether offspring receive quantitatively similar numbers of maternal microbes. We observed that H. panicea has a maternal pool of the obligate symbiont Candidatus Halichondribacter symbioticus, and that this maternal pool is provisioned proportionally to reproductive output and allometrically by offspring size. This pattern was not observed for the total bacterial community. Experimental perturbation by antibiotics could not reduce the abundance of Ca. H. symbioticus in larvae, while the total bacterial community could be reduced without affecting the ability of larvae to undergo metamorphosis. A trade-off between offspring size and number is, by definition, maternal provisioning and parallel differences in Ca. H. symbioticus abundance would suggest that this obligate symbiont is also provisioned.
... To build an understanding of the role that microbes play in the health, and ultimately, evolution of organisms, we must determine the mechanism of intergenerational transfer and maintenance of microbial members of the holobiont. While there are examples of vertical transmission of obligate symbionts in some bivalves [18,19], it is unknown how the complex communities of host microbes are transferred between generations and throughout different life cycle stages. Oysters are an excellent model system to study the role of horizontal and vertical transmission in marine invertebrates. ...
Background
The term holobiont is widely accepted to describe animal hosts and their associated microorganisms. The genomes of all that the holobiont encompasses, are termed the hologenome and it has been proposed as a unit of selection in evolution. To demonstrate that natural selection acts on the hologenome, a significant portion of the associated microbial genomes should be transferred between generations. Using the Sydney Rock Oyster ( Saccostrea glomerata ) as a model, we tested if the microbes of this broadcast spawning species could be passed down to the next generation by conducting single parent crosses and tracking the microbiome from parent to offspring and throughout early larval stages using 16S rRNA gene amplicon sequencing. From each cross, we sampled adult tissues (mantle, gill, stomach, gonad, eggs or sperm), larvae (D-veliger, umbo, eyed pediveliger, and spat), and the surrounding environment (water and algae feed) for microbial community analysis.
Results
We found that each larval stage has a distinct microbiome that is partially influenced by their parental microbiome, particularly the maternal egg microbiome. We also demonstrate the presence of core microbes that are consistent across all families, persist throughout early life stages (from eggs to spat), and are not detected in the microbiomes of the surrounding environment. In addition to the core microbiomes that span all life cycle stages, there is also evidence of environmentally acquired microbial communities, with earlier larval stages (D-veliger and umbo), more influenced by seawater microbiomes, and later larval stages (eyed pediveliger and spat) dominated by microbial members that are specific to oysters and not detected in the surrounding environment.
Conclusion
Our study characterized the succession of oyster larvae microbiomes from gametes to spat and tracked selected members that persisted across multiple life stages. Overall our findings suggest that both horizontal and vertical transmission routes are possible for the complex microbial communities associated with a broadcast spawning marine invertebrate. We demonstrate that not all members of oyster-associated microbiomes are governed by the same ecological dynamics, which is critical for determining what constitutes a hologenome.
... Pliocardiine clams are another group of endemic macrofauna in chemosynthesis-based ecosystems, with several populations of A. marissinica having been discovered at the Haima cold seep only Ip et al., 2021 ). Previous microscopic and molecular analyses uncovered that thiotrophic gammaproteobacterial endosymbionts of pliocardiine clams reside not only in gills, but also in ovaries and eggs, suggesting vertical transmission of endosymbionts to the offspring (i.e., maternal transmission; Cary and Giovannoni, 1993 ;Kojima and Ohta, 1997 ). This is different from bathymodioline mussels and siboglinid tubeworms, which obtain endosymbionts horizontally for each new generation from the ambient environment during their early life stage (i.e., environmental transmission; Won et al., 2003 ;Nussbaumer et al., 2006 ). ...
After nearly 20 years of exploration, various areas of the seafloor with hydrocarbon seepage have been discovered in the South China Sea (SCS). Through linking subsurface and seafloor biospheres, seeps have become natural laboratories to enhance our knowledge on the processes of hydrocarbon migration and associated hydrocarbon-based ecosystems in the marine environment. This review summarizes the distribution of macrofauna in the active seep areas of the SCS and describes the physiological characteristics of three groups of representative macrofauna (bathymodioline mussels, pliocardiine clams, siboglinid tubeworms). In addition, a comparison is made between the seep macrofauna of the SCS and the Northwest Pacific Ocean. Modes of energy transfer and microbial processes are elucidated by integrating trace element, stable isotope, and lipid biomarker data of macrofaunal soft tissues. Our aims are to provide an overview of the macro-ecology of the SCS seeps and to underscore aspects which desire more research efforts in the future.
... Similar processes occur across the animal kingdom (Bright & Bulgheresi 2010), and this includes many groups of marine invertebrates. Specifically, brittle stars (Walker & Lesser 1989), bryozoans (Lopanik et al. 2006), clams (Cary & Giovannoni 1993), corals (Mies et al. 2017), crabs (Goffredi et al. 2014), lobsters (Gil-Turnes & Fenical 1992, nudibranchs (Klussmann-Kolb & Brodie 1999), sea ur chins (Carrier et al. 2021), shrimps (Gil-Turnes et al. 1989, sponges (Maldonado 2007), and worms (Giere & Langheld 1987) all transmit microbes from mother to offspring. Therefore, if microbes are maternally provisioned, then one expectation would be that components of these symbiont communities covary with some of the ecological factors that influence maternal provisioning. ...
Mothers impact the survival and performance of their offspring through the resources they provision, and the degree of maternal investment in an individual offspring can be broadly estimated by egg size for organisms that lack parental care. Animals may also actively maintain symbiotic partnerships with microorganisms through the germ line, but whether microbes are a fundamental component of maternal provisioning is an untested hypothesis in evolutionary symbiosis. We present a preliminary test of this by comparing the egg-associated microbiota of 10 sea urchin species with ecological factors known to influence egg size. We found that the microbiota associated with sea urchin eggs had a phylogenetic signal in both composition and richness, which varied between years but not between individuals or within a clutch. Moreover, we found a negative correlation between microbiome richness and taxonomic dominance, and that community diversity covaried with egg size and energetic content but not with pelagic larval duration or latitude. These data suggest that there are multiple parallels between the ecological factors that govern changes in egg size and microbiome composition and diversity, implying that microbial symbionts may be another constituent potentially provided by the mother.
The ultrastructure of oogenic cells and mature oocytes has been described in a deep-sea clam, Calyptogena pacifica, for the first time. Specimens were collected in June 2018 from the Piip volcano in the Bering Sea (55.3821° N, 167.2613° E). Female germ cells developed in the branched gonadal tubules. The pattern of oogenic development is similar to that in littoral species. Although somatic accessory cells do not form any follicles around developing oocytes, these cells have a well-developed endoplasmic reticulum, which could be a source of yolk precursors and be involved in vitellogenesis. Lipid droplets appear in early oocytes, but they disappear later, suggesting that developing oocytes use these lipid droplets. At the onset of vitellogenesis, developing oocytes accumulate lipids in the ooplasm facing the gonadal tubule wall. Yolk granules, in contrast, appear at later stages of oogenesis. Mature oocytes, which are 200 µm in diameter, are filled with a significant volume of lipid droplets (occupying 24% of ooplasm) and a smaller volume of yolk granules (~ 8%). The mature oocyte’s features, i.e., the large cell size (~ 200 µm in diameter) and the high abundance of ooplasmic inclusions, are typical of species with lecithotrophic development. The large volume of lipid droplets would enhance buoyancy of the oocytes and provide embryos and larvae with nutrients during dispersal. Symbiotic bacteria were found in accessory cells, in vitellogenic oocytes, and within remnants of degenerating oocytes.
Hydrothermal vents were first discovered on the Galapagos Rift in 1977, and in the intervening 4 decades we have learned a great deal about these unique oases of life in the cold, dark, high pressure environments of the deep seafloor. Found on mid‐ocean ridges, back‐arc basins, and seamounts around the world, they result from the interaction of cold seawater with oceanic crust that has been superheated by underlying volcanic activity. Hydrothermal fluids leave the seafloor and form sulfide chimneys. These fluids are highly reducing, and contain toxic materials such as hydrogen sulfide, which provides the necessary components of life for the chemoautotrophic organisms forming the base of the food web in these ecosystems. Animals living at these vents have adapted to the harsh conditions by relying on chemoautotrophic microorganisms, either directly as a source of food, or in symbiotic association. They use varied strategies to maintain their symbiotic associations and to thrive at hydrothermal vents. While many animals characterize vent communities, some specific examples including tubeworms, Pompeii worms, mollusks, and crustaceans are described in more detail here. As we learn more about these widely spaced communities biogeographical patterns emerge, increasing our understanding of the evolution of vent communities.
Animal development is an inherently complex process that is regulated by highly conserved genomic networks, and the resulting phenotype may remain plastic in response to environmental signals. Despite development having been studied in a more natural setting for the past few decades, this framework often precludes the role of microbial prokaryotes in these processes. Here, we address how microbial symbioses impact animal development from the onset of gametogenesis through adulthood. We then provide a first assessment of which developmental processes may or may not be influenced by microbial symbioses and, in doing so, provide a holistic view of the budding discipline of developmental symbiosis.
It has been proposed that a bacterium isolated from the gills of shipworms (teredinid mollusks) is, by virtue of its ability both to degrade cellulose and to fix dinitrogen, the symbiont that enables these mollusks to utilize wood as their principal food source. The phylogenetic affiliation of four of these bacteria isolated from wood-boring bivalve mollusks was determined by 16S rRNA sequence analysis by using the reverse transcriptase method with six oligodeoxynucleotide primers. The four bacterial strains tested had indistinguishable 16S rRNA sequences, supporting the previous conclusion, based on phenotypic characterization, that these isolates represent a single species. Evolutionary distance matrix analysis of the RNA sequence indicated that the bacterial symbiont falls within the gamma-3 subdivision of the Proteobacteria and is distinct from other known bacterial genera. In situ localization of the bacterial symbiont in tissue sections of the shipworm Lyrodus pedicellatus was determined by using a 16S rRNA-directed oligodeoxynucleotide hybridization probe specific for the bacterium isolated from shipworm gill tissue. Fluorescence microscopy showed that the specific probe bound to L. pedicellatus tissue at sites coincident with the location of symbiont cells and that it did not bind to other host tissues. This technique provided direct visual evidence that the cellulolytic, nitrogen-fixing bacterial isolates were the symbionts observed within the gill of L. pedicellatus.
Near bottom water temperatures were mapped along 400 km of the strike of the Juan de Fuca Ridge as part of a combined Sea MARC/Seabeam experiment to image the variability of morphology and structure along a spreading center segment. The water temperature data collected by a continuously towed thermistor chain, in addition to salinity data, indicate that there are four geothermal areas spaced at distances of 100 km from each other south of the Cobb propagator and one field just to the north of the propagator on the Endeavor Ridge segment. Each thermal region is located above a morphological dome on the spreading center. These domes are an average of 100-200 m shallower than the rest of the axis. The structure of bottom water suggests that the geothermal regions are on average 20 km long and that the heat from these fields raises the temperature in the water column by a minimum of 0.06°C up to 300 m above the bottom. Two simple models are used to estimate the heat flux associated with these features.
The role of sulphide oxidation-driven production of reduced carbon in the nutrition of animals adapted to life in sulphide-rich habitats such as the deep-sea hydrothermal vents and intertidal mudflats has been a topic of recent interest1–4. Chemoautotrophic sulphide-oxidizing bacteria have been isolated from samples of sulphide-rich vent water5–8, and it has been suggested that these could provide a food source for filter-feeding animals that live at the vents. The recent discovery of prokaryotic cells9 and activities of sulphide-oxidizing enzymes (which generate reducing power and ATP) and Calvin—Benson cycle enzymes10 within the trophosome tissue of the large vestimentiferan tubeworm of the vents, Riftia pachyptila Jones (Phylum Pogonophora)11 suggests that sulphide-oxidizing chemoauto-trophic bacteria exist in a symbiotic relationship with at least this vent species. This discovery led us to measure enzyme activities associated with sulphide oxidation, the Calvin-Benson cycle and nitrate reduction in a variety of other vestimentiferan tube-worms and bivalve molluscs which occur in sulphide-rich habitats. All the vestimentiferan worms and several of the molluscs were found to contain these enzymatic activities, suggesting that the putative animal-bacterial symbiosis first described in Riftia pachyptila may be of widespread occurrence in species living in environments offering simultaneous access to sulphide and oxygen.
From differences in size and structure, it appears there are two species of gram-negative bacteria in the gutless oligochaetes Phallodrilus leukodermatus and P. planus from Bermuda. A non-random, differentiated and consisten distribution pattern of the extracellular bacteria along the length of the worm's body underlines the regulated nature of the bacterial colonization. This emerges also from studies on the transfer of the bacteria between host generations: exceptional for oligochaetes, eggs are deposited singly in a sticky mucus sheath and not together in a cocoon. They become infected immediately at oviposition, apparently by intrusion from large stores of bacteria in a genital pad abutting the female pores. During ontogenesis, a balance is established between extracellular, active bacteria and intracellular lytic forms enclosed in vacuoles by the epidermal cells. In early developmental stages, lytic bacteria prevail, but older worms harbour mainly extracellular prokaryotes underneath their cuticle. The thick epidermis/cuticle complex is differentiated in regular zones with a progressive trend towards enclosing and digesting bacteria intracellularly in the deeper layers. These are the first results on the transfer and biological fate of endosymbiotic bacteria living in animals from sulfide biotopes.
Species endemic to hydrothermal vent environments face difficult ecological and evolutionary conditions, especially since vent sites are irregularly distributed in space and ephemeral. Theoretical predictions for an optimal life history for an endemic organism may not be matched by one of the vent community's prominent species, the vestimentiferan Riftia pachyptila. Population samples were collected by submersible from vent sites along the Galapagos Rift and at 21°N along the East Pacific Rise.Allozymes were used to examine the population genetics of R. pachyptila, including the estimation of genetic variability of the species and differentiation of populations at different vent sites. For the 13 enzyme loci assayed by starch-gel electrophoresis, 31% were polymorphic. Heterozygosity was low: 1.5%. Genetic divergence between samples from the two regions was small but significant: genetic distance, Nei's D, was 0.008 and Wright's measure of variance partitioning, FST = 0.025 (p < 0.05) after correction for small sample sizes. Genotypic frequencies also provided evidence of the differentiation of populations: there was a deficiency of one class of heterozygotes (Pgm-1 98/100) in the pooled samples. The slight genetic differentiation may result from low genetic variability, which may prevent the use of allozymes as markers of gene flow. Endemic vent species may experience bottlenecks during colonization of new vent sites and extinction as vents become inactive. Low variability is a predictable outcome of repeated bouts of colonization and extinction, during which the effects of random genetic drift may rapidly decrease genetic variability.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
The use of Koch's technique to isolate bacteria in pure cultures has enabled thousands of bacterial species to be characterized. But for the many microorganisms that have never been cultivated, DNA amplification in vitro using the polymerase chain reaction is now making their genes accessible. Here we use this technique to study bacteria of the genus Holospora, which live in ciliates and whose phylogenetic relationship has remained unknown because they are impossible to cultivate. Species of Holospora are highly infectious and live in the nuclei of their specific host cells: H. elegans and H. undulata infect micronuclei of Paramecium caudatum, whereas H. obtusa infects the macronucleus in other strains of the same host species; Holospora species have a common developmental cycle. We have amplified, cloned and sequenced gene fragments encoding ribosomal RNA of H. obtusa. The phylogenetic position of H. obtusa in the alpha group of Proteobacteria was determined by 16S rRNA sequence analysis. The sequences were then used to design species- as well as genus-specific rRNA hybridization probes, which enabled us to detect and differentiate individual cells of the endosymbionts in situ. The large amount of rRNA in the cells indicates a high physiological activity of the endosymbionts in the host nuclei.
Lactococcus lactis subsp. cremoris is of considerable interest to the dairy industry, which relies upon the few available strains for the manufacture of cheddar cheese free of fermented and fruity flavors. The subspecies cremoris differs from related subspecies by the lack of a few phenotypic traits. Our purpose was to identify unique rRNA sequences that could be used to discriminate L. lactis subsp. cremoris from related subspecies. The 16S rRNAs from 13 Lactococcus strains were partially sequenced by using reverse transcriptase to identify domains unique to L. lactis subsp. cremoris. All five strains of the subspecies cremoris had a unique base sequence in a hypervariable region located 70 to 100 bases from the 5' terminus. In this region, all L. lactis subsp. lactis biovar diacetylactis strains examined had a sequence identical to that of L. lactis subsp. lactis 7962, which was different from other strains of the subspecies lactis by only one nucleotide at position 90 (Escherichia coli 16S rRNA structural model) (J. Brosius, J. L. Palmer, J. P. Kennedy, and H. F. Noller, Proc. Natl. Acad. Sci. USA 75:4801-4805, 1978). Oligonucleotide probes specific for the genus Lactococcus (212RLa) and for the subspecies cremoris (68RCa) were synthesized and evaluated by hybridization to known rRNAs as well as fixed whole cells. Efficient and specific hybridization to the genus-specific probe was observed for the 13 Lactococcus strains tested. No hybridization was seen with the control species. All five strains of the subspecies cremoris hybridized to the subspecies-specific probe.