Extremely halophilic archaeal communities are resilient to short-term entombment in halite

Some haloarchaea avoid the harsh conditions present in evaporating brines by entombment in brine inclusions within forming halite crystals, where a subset of haloarchaea survives over geological time. However, shifts in the community structure of halite-entombed archaeal communities remain poorly understood. Therefore, we analysed archaeal communities from in situ hypersaline brines collected from Trapani saltern (Sicily) and their successional changes in brines versus laboratory-grown halite over 21 weeks, using high-throughput sequencing. Haloarchaea were dominant, comprising >95% of the archaeal community. Unexpectedly, the OTU richness of the communities after 21 weeks was indistinguishable from the parent brine and overall archaeal abundance in halite showed no clear temporal trends. Furthermore, the duration of entombment was less important than the parent brine from which the halite derived in determining the community composition and relative abundances of most genera in halite-entombed communities. These results show that halite-entombed archaeal communities are resilient to entombment durations of up to 21 weeks, and that entombment in halite may be an effective survival strategy for near complete communities of haloarchaea. Additionally, the dominance of ‘halite specialists’ observed in ancient halite must occur over periods of years, rather than months, hinting at long-term successional dynamics in this environment.     

Halophilic Archaea cultured from ancient halite,Death Valley,California

Halophilic Archaea cultured from ancient fluid inclusions in a 90‐m‐long (0‐ to 100 000‐year‐old) salt core from Death Valley, California, demonstrate survival of bacterial cells in subsurface halite for up to 34 000 years. Five enrichment cultures, representing three genera of halophilic Archaea (Halorubrum, Natronomonas and Haloterrigena), were obtained from five surface‐sterilized halite crystals exclusively in one section of the core (13.0–17.8 m; 22 000–34 000 years old) containing perennial saline lake deposits. Prokaryote cells were observed microscopically in situ within fluid inclusions from every layer that produced culturable cells. Another 876 crystals analysed from depths of 8.1–86.7 m (10 000–100 000 years old) failed to yield live halophilic Archaea. Considering the number of halite crystals tested (culturing success of 0.6%), microbial survival in fluid inclusions in halite is rare and related to the paleoenvironment, which controls the distribution and abundance of trapped microorganisms. Two cultures from two crystals at 17.8 m that yielded identical 16S rRNA sequences (genus: Haloterrigena) demonstrate intra‐laboratory reproducibility. Inter‐laboratory reproducibility is shown by two halophilic Archaea (genus: Natronomonas), with 99.3% similarity of 16S rRNA sequences, cultured from the same core interval, but at separate laboratories.     

Reconstruction of Late Cretaceous coastal paleotemperature from halite deposits of the Late Cretaceous Nongbok Formation (Khorat Plateau,Laos)

Cretaceous evaporites of the Maha Sarakhan Formation in Thailand (e.g., the Nongbok Formation, Laos) have been studied for almost a century as the huge potash deposits in the world. The consistently high local paleotemperatures should lead to huge salt deposits during the evaporation process. Primary fluid inclusions in halite can provide surface brine water temperatures directly and quantitatively. Until now, there have been no data published from paleotemperature of primary fluid inclusions of Cretaceous halite. The non-marine halite from the Cretaceous Nongbok Formation (Laos) precipitated from shallow brine waters with temperatures of 17.7–42.3 °C.     

Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)

The Salar Grande in the Coastal Range of Northern Chile is a fossil evaporitic basin filled with almost pure halite (95% NaCl average). It is assumed that the basin has not received input of brines since the Pliocene (5.3 to 1.8 million years). Below 1 m the halite has remained undissolved since this time, whereas the upper layer has been dissolved and recrystallized by dripping fogs and occasional rainfall. We compared the archaeal community at different depths using both nested PCR and cultivation. The upper 10 cm of halite crust contained diverse haloarchaeal species, including several from new genera, but their provenance is unknown. For samples deeper in the core, a new and rigorous procedure for chemically sterilizing the surface of single halite crystals was developed. These halite crystals contained only species of the genus Halobacterium (Hbt.). Halobacterium salinarum-like sequences were detected by PCR, and evidence that they were from ancient DNA include: comparison with numerous negative controls; detection of 16S rRNA sequence differences in non-conserved regions, indicating genuine evolutionary mutations rather than PCR-cloning artefacts; independent isolation of Hbt. salinarum from ancient halite; and diverse mechanisms possessed by this species for minimizing radiation damage and thus enhancing its potential for long-term survival. Haloarchaea related to Hbt. noricense were obtained from enrichment cultures from ≈ 0.4 and 15.4 m depth. We investigated Hbt. noricense strain A1 and found that when trapped inside halite crystals its recovery was as rapid after 27 months of entombment as at day 0, faring much better than other extreme halophiles. A biogeographical investigation showed that Hbt. noricense-like organisms were: commonly found in surface-sterilized ancient halite, associated with salt mines, in halite crusts, and, despite a much more intense search, only rarely detected in surface environments. We conclude that some Halobacterium species are specialists at long-term survival in halite.     

江苏金坛盐矿形成时期盐湖水体成分:来自石盐包裹体的证据

我国白垩纪晚期-古近纪由于燕山运动和喜马拉雅运动,形成了一系列的断陷盆地,金坛盐矿就是该时期始新世中晚期的盐湖沉积.本次研究首次在国内半定量的进行了石盐原生包裹体的分析,发现其中Ca2+缺失而富含SO2-4,原始卤水成分为Na-Mg-K-Ca-C1-SO4体系.     

Characterization of a Neochlamydia-like bacterium associated with epitheliocystis in cultured Arctic charr Salvelinus alpinus

Infections of branchial epithelium by intracellular gram-negative bacteria, termed epitheliocystis, have limited culture of Arctic charr Salvelinus alpinus. To characterize a bacterium associated with epitheliocystis in cultured charr, gills were sampled for histopathologic examination, conventional and immunoelectron microscopy, in situ hybridization, 16S ribosomal DNA (rDNA) amplification, sequence analysis and phylogenetic inference. Sampling was conducted at the Freshwater Institute (Shepherdstown, West Virginia, USA) during outbreaks of epitheliocystis in April and May 2002. Granular, basophilic, cytoplasmic inclusions in charr gill were shown to stain with Macchiavello, Lendrum's phloxine-tartrazine and Gimenez histochemical techniques. Ultrastructurally, inclusions were membrane-bound and contained round to elongate reticulate bodies that were immunoreactive to an antibody against chlamydial lipopolysaccharide, suggesting the presence of similar epitopes. DNA extracted from gills supported amplification of the most polymorphic and phylogenetically relevant region of the 16S rRNA gene, which had 97 to 100% identity with several uncultured clinical Neochlamydia spp. (order Chlamydiales) Clones WB13 (AY225593.1) and WB258 (AY225594.1). Sequence-specific riboprobes localized to inclusions during in situ hybridization experiments. Taxonomic affiliation was inferred by distance- and parsimony-based phylogenetic analyses of the 16S sequence, which branched with Neochlamydia hartmannellae in the order Chlamydiales with high confidence. This is the first molecular characterization of a chlamydia associated with epitheliocystis in Arctic charr and the fourth Neochlamydia spp. sequence to be associated with epitheliocystis. Presence of a clinical neochlamydial sequence, first identified from a cat, in Arctic charr suggests a possible mammalian and piscine host range for some environmental chlamydiae.     

Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction

Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.     

Haloarchaeal diversity in 23, 121 and 419 MYA salts

DNA was extracted from surface-sterilized salt of different geological ages (23, 121, 419 million years of age, MYA) to investigate haloarchaeal diversity. Only Haloarcula and Halorubrum DNA was found in 23 MYA salt. Older crystals contained unclassified groups and Halobacterium . The older crystals yielded a unique 55-bp insert within the 16S rRNA V2 region. The secondary structure of the V2 region completely differed from that in haloarchaea of modern environments. The DNA demonstrates that unknown haloarchaea and the Halobacterium were key components in ancient hypersaline environments. Halorubrum and Haloarcula appear to be a dominant group in relatively modern hypersaline habitats.     

Halophilic archaea in the human intestinal mucosa

The human gastrointestinal tract microbiota, despite its key roles in health and disease, remains a diverse, variable and poorly understood entity. Current surveys reveal a multitude of undefined bacterial taxa and a low diversity of methanogenic archaea. In an analysis of the microbiota in colonic mucosal biopsies from patients with inflammatory bowel disease we found 16S rDNA sequences representing a phylogenetically rich diversity of halophilic archaea from the Halobacteriaceae (haloarchaea), including novel phylotypes. As the human colon is not considered a salty environment and haloarchaea are described as extreme halophiles, we evaluated and further discarded the possibility that these sequences originated from pre‐colonoscopy saline lavage solutions. Furthermore, aerobic enrichment cultures prepared from a patient biopsy at low salinity (2.5% NaCl) yielded haloarchaeal sequence types. Microscopic observation after fluorescence in situ hybridization provided evidence of the presence of viable archaeal cells in these cultures. These results prove the survival of haloarchaea in the digestive system and suggest that they may be members of the mucosal microbiota, even if present in low numbers in comparison with methanogenic archaea. Investigation of a potential physiological basis of this association may lead to new insights into gastrointestinal health and disease.     

Spherical particles of halophilic archaea correlate with exposure to low water activity - implications for microbial survival in fluid inclusions of ancient halite

Viable extremely halophilic archaea (haloarchaea) have been isolated from million‐year‐old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000‐ to 34 000‐year‐old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440–454). Searching for a method to produce such particles in the laboratory, we exposed rod‐shaped cells of Halobacterium species to reduced external water activity (a_w). Gradual formation of spheres of about 0.4 μm diameter occurred in 4 m NaCl buffer of a_w ≤ 0.75, but exposure to buffered 4 m LiCl (a_w ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re‐grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50‐fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering a_w should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.     

Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt

Prokaryotic diversity in Alpine salt sediments was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA genes, sequencing of cloned products, and comparisons with culturable strains. DNA was extracted from the residue following filtration of dissolved Permo-Triassic rock salt. Fifty-four haloarchaeal sequences were obtained, which could be grouped into at least five distinct clusters. Similarity values of three clusters to known 16S rRNA genes were less than 90%-95%, suggesting the presence of uncultured novel taxa; two clusters were 98% and 99% similar to isolates from Permo-Triassic or Miocene salt from England and Poland, and to Halobacterium salinarum, respectively. Some rock salt samples, including drilling cores, yielded no amplifiable DNA and no cells or only a few culturable cells. This result suggested a variable distribution of haloarchaea within different strata, probably consistent with the known geologic heterogeneity of Alpine salt deposits. We recently reported identical culturable Halococcus salifodinae strains in Permo-Triassic salt sediments from England, Germany, and Austria; together with the data presented here, those results suggest one plausible scenario to be an ancient continuous hypersaline ocean (Zechstein sea) populated by haloarchaea, whose descendants are found today in the salt sediments. The novelty of the sequences also suggested avoidance of haloarchaeal contaminants during our isolation of strains, preparation of DNA, and PCR reactions.     

Cloning and sequencing of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC)

AIMS: To clone and sequence the 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: The primer sets for 16S rDNA and 16S-23S rDNA ISR amplified almost the full length of 16S rDNA and 16S-23S rDNA ISR. About 1500 bp for 16S rDNA and about 720 bp for 16S-23S rDNA ISR of the rrn operon of four strains of UPTC were identified after molecular cloning and sequencing. CONCLUSIONS: The four strains and CCUG18267 of UPTC showed approximately 99% sequence homology of 16S rDNA to each other, 96-97% to Camp. coli, 97-98% to Camp. jejuni and 97-98% to Camp. lari. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the nucleotide sequence of 16S-23S rDNA ISR of UPTC has been analysed. The sequence of ISR was almost identical among the four strains of UPTC. It is interesting that the UPTC intercistronic tRNAs demonstrated an order of tRNA of 5'-16S-tRNAAla-tRNAIle-23S-3' in the organisms.     

共存于厌氧真菌分离培养液中瘤胃甲烷菌的检测及其多样性分析

对分离自山羊瘤胃的真菌分离培养液中甲烷菌进行16SrDNA扩增、DGGE分析、RFLP及测序分析,研究共存于真菌分离培养液中甲烷菌的种类及其多样性。DGGE结果显示:从厌氧真菌分离至第45代,甲烷菌多样性指数由1·32降至0·99,相似性最低为34·7%;第45代至62代,多样性指数由0·99升至1·15,相似性最低为89·2%。RFLP多态性分析69个克隆共得到5个操作分类单元,选择其中6个具有代表性的序列进行测序。序列及系统进化分析表明,属于其中3个操作分类单元的克隆最相似菌都是UnculturedarchaealsymbiontPA202,相似性均为95%,没有与这些克隆相似性较高的已培养甲烷菌;属于另外2个操作分类单元的克隆最相似菌都是Unculturedrumenmethanogen956,相似性均为97%,最相似已知菌为Methanobrevibactersp.NT7,相似性为97%。结果表明,真菌培养液中存在目前尚未分离培养的瘤胃甲烷菌。     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

Putative Phage Hyperparasite in the Rickettsial Pathogen of Abalone, “Candidatus Xenohaliotis californiensis”

Studies on the ecology of microbial parasites and their hosts are predicated on understanding the assemblage of and relationship among the species present. Changes in organismal morphology and physiology can have profound effects on host–parasite interactions and associated microbial community structure. The marine rickettsial organism, “Candidatus Xenohaliotis californiensis” (WS-RLO), that causes withering syndrome of abalones has had a consistent morphology based on light and electron microscopy. However, a morphological variant of the WS-RLO has recently been observed infecting red abalone from California. We used light and electron microscopy, in situ hybridization and16S rDNA sequence analysis to compare the WS-RLO and the morphologically distinct RLO variant (RLOv). The WS-RLO forms oblong inclusions within the abalone posterior esophagus (PE) and digestive gland (DG) tissues that contain small rod-shaped bacteria; individual bacteria within the light purple inclusions upon hematoxylin and eosin staining cannot be discerned by light microscopy. Like the WS-RLO, the RLOv forms oblong inclusions in the PE and DG but contain large, pleomorphic bacteria that stain dark navy blue with hematoxylin and eosin. Transmission electron microscopy (TEM) examination revealed that the large pleomorphic bacteria within RLOv inclusions were infected with a spherical to icosahedral-shaped putative phage hyperparasite. TEM also revealed the presence of rod-shaped bacteria along the periphery of the RLOv inclusions that were morphologically indistinguishable from the WS-RLO. Binding of the WS-RLO-specific in situ hybridization probe to the RLOv inclusions demonstrated sequence similarity between these RLOs. In addition, sequence analysis revealed 98.9–99.4 % similarity between 16S rDNA sequences of the WS-RLO and RLOv. Collectively, these data suggest that both of these RLOs infecting California abalone are “Candidatus Xenohaliotis californiensis,” and that the novel variant is infected by a putative phage hyperparasite that induced morphological variation of its RLO host.     

Life at low water activity

Two major types of environment provide habitats for the most xerophilic organisms known: foods preserved by some form of dehydration or enhanced sugar levels, and hypersaline sites where water availability is limited by a high concentration of salts (usually NaCl). These environments are essentially microbial habitats, with high-sugar foods being dominated by xerophilic (sometimes called osmophilic) filamentous fungi and yeasts, some of which are capable of growth at a water activity (a(w)) of 0.61, the lowest a(w) value for growth recorded to date. By contrast, high-salt environments are almost exclusively populated by prokaryotes, notably the haloarchaea, capable of growing in saturated NaCl (a(w) 0.75). Different strategies are employed for combating the osmotic stress imposed by high levels of solutes in the environment. Eukaryotes and most prokaryotes synthesize or accumulate organic so-called 'compatible solutes' (osmolytes) that have counterbalancing osmotic potential. A restricted range of bacteria and the haloarchaea counterbalance osmotic stress imposed by NaCl by accumulating equivalent amounts of KCl. Haloarchaea become entrapped and survive for long periods inside halite (NaCl) crystals. They are also found in ancient subterranean halite (NaCl) deposits, leading to speculation about survival over geological time periods.     

MOLECULAR IDENTIFICATION OF A BACTERIUM ASSOCIATED WITH GALL FORMATION IN THE MARINE RED ALGA PRIONITIS LANCEOLATA1

The polymerase chain reaction (PCR) was used to amplify eubacterial small-subunit (16S) ribosomal DNA (rDNA) genes from galls of the marine red alga Prionitis lanceolata Harvey (Gigartinales). These tumors consist of hypertrophied algal cells containing large numbers of intercellular bacteria that remain uncultivable. PCR-amplified 16S rDNAs from surface-sterilized gall tissue plugs were cloned, sequenced, and analyzed by alignment to available small-subunit rRNA sequences (University of Illinois Ribosomal Database Project). Variable regions were identified and used to construct a fluorescently labeled, species-specific oligodeoxynucleotide probe for whole cell in situ hybridization to the gall symbiont. Probe 949 (PLANC.949) localized the P. lanceolata bacterial symbiont in preparations from mature gall tissue. This probe did not hybridize to the rDNA of closely related bacteria included as controls in the same hybridization reactions, In situ hybridization revealed the presence of the same bacterium in association with P. lanceolata gall formation from three central California localities. Distance and parsimony analyses suggest that this organism is a member of the Proteobacteria (alpha subdivision; Rhodobacter group) and is most closely related to Roseobacter denitrificans .     

Evaluation of a plasmid-based 16S-23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater

A plasmid-based 16S-23S rDNA intergenic spacer region (ISR) array was developed and optimized for analysis of microbial diversity within complex environmental samples. Plasmid probes with 16S-23S rDNA ISR inserts (800-1500 bp) from industrial wastewater treatment plant (WWTP) microorganisms were arrayed onto glass slides. Hybridization of fluorescently labeled target sequences from two clones from the ISR WWTP library to arrayed probes showed that there was a good linear relationship between hybridization intensity and ISR similarity (r(2)=0.82). Hybridization was highly specific (average background from arrayed probes with less than 80% similarity in ISR sequence was less than 7%). Strong fluorescence intensity corresponded to near-perfect match clones (99% or greater similarity in ISR sequence). A majority of probes (79%) showed no background hybridization. However, weak background (less than 50% for arrayed probes with 90% and 95% similarity in the 16S rRNA genes) was observed from closely related microorganisms. Background fluorescence from the negative control (plasmid vector with no insert) was similar to water and dimethyl sulfoxide (DMSO)-negative controls. Hybridization using fluorescently labeled ISR sequences from a mixed community sample produced strong fluorescent signals with no background from negative controls. A Cy5-labeled reference standard, part of the vector and present in every spotted probe, was used to normalize hybridization values. These results indicate that arrayed plasmid containing ISR probe insert sequences provides specificity and sensitivity for microbial community analysis in a high-throughput array format.     

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA–DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.     

Two Intracellular Symbiotic Bacteria from the Mulberry Psyllid Anomoneura mori (Insecta, Homoptera)

We characterized the intracellular symbiotic bacteria of the mulberry psyllid Anomoneura mori by performing a molecular phylogenetic analysis combined with in situ hybridization. In its abdomen, the psyllid has a large, yellow, bilobed mycetome (or bacteriome) which consists of many round uninucleated mycetocytes (or bacteriocytes) enclosing syncytial tissue. The mycetocytes and syncytium harbor specific intracellular bacteria, the X-symbionts and Y-symbionts, respectively. Almost the entire length of the bacterial 16S ribosomal DNA (rDNA) was amplified and cloned from the whole DNA of A. mori, and two clones, the A-type and B-type clones, were identified by restriction fragment length polymorphism analysis. In situ hybridization with specific oligonucleotide probes demonstrated that the A-type and B-type 16S rDNAs were derived from the X-symbionts and Y-symbionts, respectively. Molecular phylogenetic analyses of the 16S rDNA sequences showed that these symbionts belong to distinct lineages in the γ subdivision of the Proteobacteria. No 16S rDNA sequences in the databases were closely related to the 16S rDNA sequences of the X- and Y-symbionts. However, the sequences that were relatively closely related to them were the sequences of endosymbionts of other insects. The nucleotide compositions of the 16S rDNAs of the X- and Y-symbionts were highly AT biased, and the sequence of the X-symbiont was the most AT-rich bacterial 16S rDNA sequence reported so far.