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PubMed1996

Genome segment 5 of rice ragged stunt virus encodes a virion protein.

The complete nucleotide sequence of the genome segment 5 (S5) of a Thai isolate of rice ragged stunt virus (RRSV) was determined. The 2682 nucleotide sequence contains a single long open reading frame capable of encoding a polypeptide with a molecular mass of approximately 91 kDa. Polypeptides encoded by various truncated cDNAs of S5 were expressed using the pGEX fusion protein vector and the highest level of fusion protein was obtained from a construct encoding a hydrophilic region of S5 protein. Antibodies raised against this fusion protein recognized a minor polypeptide, with a molecular mass of approximately 91 kDa, that was present in purified preparations of RRSV particles, infected insect vectors and infected rice plants. This indicates that RRSV S5 encodes a minor structural protein. Comparing the RRSV S5 sequence with sequences of other reoviruses did not reveal any significant sequence similarities.

Amino Acid Sequence↗
PubMed1997

The proteins encoded by rice grassy stunt virus RNA5 and RNA6 are only distantly related to the corresponding proteins of other members of the genus Tenuivirus.

The genome of rice grassy stunt virus (RGSV) consists of six RNA segments. The nucleotide (nt) sequences of the two smallest segments, RNAs 5 and 6, were determined and found to comprise 2704 and 2584 nt, respectively. The 5'- and 3'-terminal sequences of both RNAs were identical over a length of 21 nt and could potentially form a panhandle-like structure due to intramolecular complementarity. Each RNA segment contained a virus (v) sense open reading frame (ORF) in the 5'-proximate region, and a virus complementary (vc) ORF in the 3'-proximate region, indicating an ambisense coding strategy. The protein encoded by the ORF on the vc strand of RNA5 was identified as the viral nucleocapsid protein (M(r) 35927). The ORF on the v strand of RNA6 encoded a protein of M(r) 20581 which represented the major nonstructural protein, previously shown to be produced in RGSV-infected rice tissues. The predicted proteins encoded by RGSV RNAs 5 and 6 were only distantly similar in sequence to the four proteins encoded by RNAs 3 and 4 of other viruses belonging to the genus Tenuivirus. These low sequence similarities, together with the apparently distinct number of genome segments, set RGSV apart from the other tenuiviruses and indicate that it should be placed in a taxonomically separate genus.

Amino Acid Sequence↗
PubMed1998

Ten distinct circular ssDNA components, four of which encode putative replication-associated proteins, are associated with the faba bean necrotic yellows virus genome.

Four further circular ssDNA components (C7-C10), about 1 kb in size and structurally similar to the previously described components (C1-C6) found associated with a Syrian (Sy) isolate of faba bean necrotic yellows virus (FBNYV), have been identified. Similar to C1 and C2, two of the new components (C7 and C9) encode putative replication-associated (Rep) proteins of 33.2 and 32.7 kDa, respectively, the former of which is 90% identical to the C10 Rep protein of milk vetch dwarf virus (MDV). C8 encodes a putative protein (17.4 kDa) whose function is unknown, but which is highly conserved between FBNYV and the other nanoviruses MDV, subterranean clover stunt virus and banana bunchy top virus. The putative protein (19.7 kDa) encoded by C10 contains an LXCXE motif, which is also present in the homologues of its relatives, suggesting that they may all interact with plant retinoblastoma-like proteins. Sequence information for seven components of an Egyptian (Eg) FBNYV isolate indicated that six of them share > 96% identity with FBNYV-Sy. However, the C1 Rep protein of FBNYV-Eg was only 63.5% identical to that of FBNYV-Sy, but was 88.3 % identical to the MDV-C2 Rep protein. We conclude that the FBNYV genome consists of seven to ten components, six of which encode non-Rep proteins. All ten components of the FBNYV genome, except the Rep components C2 and C9, had closely related counterparts in the MDV genome. In spite of this similarity, FBNYV and MDV appear to be distinct virus species.

Amino Acid Sequence↗
PubMed1998

Restricted expression of Epstein-Barr virus (EBV)-encoded, growth transformation-associated antigens in an EBV- and human herpesvirus type 8-carrying body cavity lymphoma line.

A body cavity lymphoma-derived cell line (BC1), known to carry both Epstein-Barr virus (EBV) and human herpes virus type 8 (HHV-8; or Kaposi's sarcoma-associated herpesvirus, KSHV), was analysed for the expression of EBV-encoded, growth transformation-associated antigens and cellular phenotype by immunofluorescence staining, Western blotting, RT-PCR and flow cytometry. A similar phenotypic analysis was also performed on another body cavity lymphoma line, BCBL1, that is singly infected with HHV-8. Phenotypically, the two lines were closely similar. Although both lines are known to carry rearranged immunoglobulin genes, they were mostly negative for B-cell surface markers. Both expressed the HHV-8-encoded nuclear antigen (LNA1). Similarly to Epstein-Barr nuclear antigen type 1 (EBNA1), LNA1 was associated with the chromatin in interphase nuclei and the mitotic chromosomes in metaphase. It accumulated in a few well-circumscribed nuclear bodies that did not co-localize with EBNA1. BC1 cells expressed EBNA1, LMP2A and EBV-encoded small RNAs but not EBNA2-6, LMP1 and LMP2B. They were thus similar to type I Burkitt's lymphoma cells and latently infected peripheral B-cells. Analysis of the splicing pattern of the EBNA1-encoding message by RT-PCR showed that BC1 cells used the QUK but not the YUK splice, indicating that the mRNA was initiated from Qp and not from Cp or Wp.

Antigens, Viral↗
PubMed1999

Detection and characterization of proteins encoded by the second ORF of the M2 gene of pneumoviruses.

The nucleotide sequence of the M2 gene of pneumonia virus of mice (PVM) was determined. The sequence showed that the gene encoded a protein of 176 amino acids with a predicted molecular mass of 20165 Da from a major ORF, which is smaller than the equivalent proteins encoded by human, bovine and ovine respiratory syncytial (RS) viruses. The PVM M2 protein is conserved, having 41% similarity to the equivalent human RS virus protein. In common with the M2 genes of the RS viruses and avian pneumovirus (APV), the PVM mRNA also contained a second ORF (ORF2) that partially overlaps the first ORF and which is capable of encoding a 98 residue polypeptide. No significant sequence identity could be detected between the putative M2 ORF2 proteins of PVM, APV and the RS viruses. The expression of the M2 ORF2 proteins of the pneumoviruses was investigated by using monospecific antisera raised against GST fusion proteins. Western blot analysis demonstrated the presence of polypeptides encoded by M2 ORF2 of PVM and RS virus corresponding with those predicted by in vitro translation studies, but this was not the case for APV. The PVM polypeptide was present as three distinct products in vivo. The PVM and RS virus polypeptides were also detected in cells by immunofluorescence, which showed that both were present in the cytoplasm with a degree of localization in inclusion bodies. No APV M2 ORF2 protein could be detected in vivo. The RS virus M2 ORF2 polypeptide was shown to accumulate during infection and the potential implications of this are discussed.

Amino Acid Sequence↗
PubMed2001

Banana bunchy top nanovirus DNA-1 encodes the 'master' replication initiation protein.

Banana bunchy top nanovirus has a multicomponent, circular single-stranded DNA genome comprising at least six integral components, BBTV DNA-1 to -6, which have been consistently associated with bunchy top disease worldwide. At least three other components, BBTV S1, S2 and Y, which have been isolated from Taiwanese BBTV isolates, do not appear to be integral components. We show here that both BBTV DNA-1 and S1, which encode replication initiation (Rep) proteins, were capable of self-replication when bombarded into banana embryogenic cell suspensions. However, only BBTV DNA-1 was capable of directing the replication of two other BBTV genomic components, namely BBTV DNA-3 which encodes the coat protein, and DNA-5 which encodes a retinoblastoma binding-like protein. These results indicate that (i) BBTV DNA-1 is the minimal replicative unit of BBTV and encodes the 'master' viral Rep and (ii) BBTV S1 is possibly a satellite DNA which is unable to replicate integral BBTV components.

Biolistics↗
PubMed2001

Characterization and functional analysis of Serp3: a novel myxoma virus-encoded serpin involved in virulence.

Myxoma virus (MV), a member of the family Poxviridae, is the causative agent of myxomatosis, a fatal disease of the European rabbit. The MV genome is a linear, double-stranded DNA molecule that encodes several factors important for evasion of the host immune system. Sequencing the right-end region of the MV genome identified an 801 bp open reading frame (ORF) encoding a polypeptide that belongs to the serpin superfamily. To date, two MV-encoded serpins have been characterized: SERP-1 binds to several targets and is an anti-inflammatory molecule, whereas Serp2 is essential for virus virulence and has both anti-inflammatory and anti-apoptotic effects. Thus, Serp3 is the third MV-encoded serpin. DNA sequence analysis of Serp3 indicated a similarity to poxvirus late promoters, which was confirmed by mRNA expression analysis. Serp3 has an atypical serpin motif and has significant sequence deletions as compared to most cellular and viral serpins. However, molecular modelling studies suggested that Serp3 can retain the overall serpin fold. Insertional inactivation of the serp3 ORF led to a significant attenuation of virulence in vivo (as measured by the increase in survival of infected rabbits) and limited dissemination of the virus to secondary sites of infection. In rabbits infected with a Serp3 deletion mutant (MV-Serp3(-)), the main histopathological feature is the absence of secondary myxomas. Both wild-type MV and MV-Serp3(-) replicate at comparable levels in vivo. Serp3 may represent a significant virulence factor of MV and probably acts in synergy with other viral proteins.

Amino Acid Motifs↗
PubMed1986

Effects of immune colostrum on the expression of a K88 plasmid encoded determinant: role of plasmid stability and influence of phenotypic expression of K88 fimbriae.

Passaging of the K88-positive Escherichia coli strain CN6913 through synthetic medium containing immune colostrum gave rise to large numbers of K88-negative CN6913 variants. These K88-negative variants had all lost a single large plasmid known to encode the K88 genetic determinant. Four other large plasmids harboured by this strain were unaffected. Viable K88-positive and K88-negative variants of CN6913 accumulated at a similar rate in synthetic medium and in medium containing non-immune colostrum. In the presence of immune colostrum, viable cells of the K88-negative variant accumulated faster and to a greater extent in cultures than the K88-positive variant if incubated at 37 degrees C, which favours the phenotypic expression of K88. However, when similar cultures were incubated at 18 degrees C, a temperature known to inhibit phenotypic expression of K88, the accumulation of viable cells of the two variants was strictly comparable in all media and no loss of plasmid or increase in K88-negative variants was observed. Cells containing a pBR322-based K88-encoding recombinant plasmid were also eliminated by immune colostrum whereas cells containing pBR322 were not. Plasmids encoding the K99 antigen were not readily eliminated from strains passaged through medium containing immune colostrum. K99-negative variants that were detected still harboured the K99-encoding plasmid.

Animals↗
PubMed1987

Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes.

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.

Bacterial Toxins↗
PubMed1989

Comparison of Vero-cytotoxin-encoding phages from Escherichia coli of human and bovine origin.

Phages encoding production of Vero cytotoxins VT1 or VT2 were isolated from strains of Escherichia coli of human and bovine origin. Two human strains of serotype O157: H7 produced both VT1 and VT2 and each carried two separate phages encoding either VT1 or VT2. The phages were morphologically similar to each other and to a VT2 phage previously isolated from a strain of serotype O157: H-; all had regular hexagonal heads and short tails. The phages had similar genome sizes and DNA hybridization and restriction enzyme digestion showed that the DNAs were very closely related. This contrasts with another report that one of the strains tested (933) released two clearly distinguishable phages separately encoding VT1 and VT2. The O157 phages differed from a VT1 phage isolated from a bovine E. coli strain belonging to serotype O26: H11 and from the reference VT1 phage isolated previously from a human strain, H19, of serotype O26: H11. The two O26 phages were morphologically similar with elongated heads and long tails. They had similar genome sizes and DNA hybridization indicated a high level of homology between them. Hybridization of an O157 phage DNA probe to DNA of the O26 phages, and vice versa, showed there was some cross-hybridization between the two types of phage. A phage from a bovine strain of serotype O29: H34 had a regular hexagonal head and short tail resembling those of the O157 phages. The DNA was distinguishable from that of all the other phages tested in restriction digest patterns but hybridized significantly to that of an O157 phage. Hybridization of the phage genomes with VT1 and VT2 gene probes showed that sequences encoding these toxins were highly conserved in the different phages from strains belonging to the three serogroups.

Animals↗
PubMed1991

Isolation and characterization of cDNA clones encoding polypeptides related to a Dictyostelium discoideum cyclic AMP binding protein.

By screening a cDNA library with a cDNA encoding the Dictyostelium discoideum cAMP-binding protein CABP1, under conditions of reduced stringency, we have isolated clones which code for two closely related molecules. Hybrid selection experiments indicated that these cDNAs encoded polypeptides with molecular masses of 34 (p34) and 31 (p31) kDa, both of which were recognized by anti-CABP1 monoclonal antibodies. Sequence analysis revealed that the clones were identical except for the presence of a 102 nucleotide segment inserted in-frame in the p34 cDNAs, just downstream of the translation initiation codon. DNA blot analysis suggested that p34 and p31 were encoded by the same gene. This hypothesis was strongly supported by the observation that both polypeptides were generated when a single cDNA was expressed under the control of the actin 15 promoter in D. discoideum cells. RNA blot analysis indicated that the cDNAs were complementary to three developmentally regulated transcripts of sizes 1.15 kb, 1.25 kb and 1.4 kb. Comparison of the derived amino acid sequences of p34 and p31 with those of the two subunits of CABP1 indicated that these polypeptides were very closely related, and that the corresponding genes probably arose by duplication followed by sequence divergence. Finally, the carboxy termini of these four polypeptides demonstrated 50% similarity to two polypeptides encoded by a bacterial plasmid which confers resistance to tellurium anions.

Actins↗
PubMed1997

Functional and genetic characterization of mcpC, which encodes a third methyl-accepting chemotaxis protein in Bacillus subtilis.

A 3135 bp DNA segment downstream of the spl gene on the Bacillus subtilis chromosome was cloned and its nucleotide sequence determined. An open reading frame capable of encoding a putative protein of 654 amino acids with a calculated molecular mass of 72.1 kDa was identified. The deduced amino acid sequence was similar to the McpA and McpB proteins of B. subtilis. McpA and McpB encode different methyl-accepting chemotaxis proteins (MCPs). A mutant strain containing an antibiotic resistance DNA cassette inserted into the region containing the MCP-like reading frame suffered a complete loss of taxis to the amino acids cysteine, proline, threonine, glycine, serine, lysine, valine and arginine. The open reading frame was designated mcpC. The wild-type and an mcpC mutant strain were analysed for their content of methylated proteins and it was found that mcpC encodes a methylated membrane protein that has previously been designated H3. These results show that mcpC encodes a third MCP in B. subtilis. The transcription start site upstream of the mcpC gene was determined by primer extension analysis and it was found to be preceded by a potential promoter sequence that is recognized by the sigma D form of RNA polymerase. The level of beta-galactosidase expressed from a transcriptional mcpC-lacZ fusion was increased threefold when cells entered the stationary phase. No beta-galactosidase could be detected in a sigD genetic background.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of a gene (LIP1) which encodes a lipase from the pathogenic yeast Candida albicans.

Extracellular phospholipases are demonstrated virulence factors for a number of pathogenic microbes. The opportunistic pathogen Candida albicans is known to secrete phospholipases and these have been correlated with strain virulence. In an attempt to clone C. albicans genes encoding secreted phospholipases, Saccharomyces cerevisiae was transformed with a C. albicans genomic library and screened for lipolytic activity on egg-yolk agar plates, a traditional screen for phospholipase activity. Two identical clones were obtained which exhibited lipolytic activity. Nucleotide sequence analysis identified an ORF encoding a protein of 351 amino acid residues. Although no extensive homologies were identified, the sequence contained the Gly-X-Ser-X-Gly motif found in prokaryotic and eukaryotic lipases, suggesting a similar activity for the encoded protein. Indeed, culture supernatants from complemented yeast cells contained abundant hydrolytic activity against a triglyceride substrate and had no phospholipase activity. The data suggest that C. albicans, in addition to phospholipases, also has lipases. Southern blot analyses revealed that C. albicans may contain a lipase gene (LIP) family, and that a lipase gene(s) may be present in Candida parapsilosis, Candida tropicalis and Candida krusei, but not in Candida pseudotropicalis, Candida glabrata or S. cerevisiae. Northern blot analyses showed that expression of the LIP1 transcript, the cloned gene which encodes a lipase, was detected only when C. albicans was grown in media containing Tween 80, other Tweens or triglycerides as the sole carbon source, and not in Sabouraud Dextrose Broth or yeast/peptone/dextrose media. Additionally, carbohydrate supplementation inhibited LIP1 expression. Cloning this gene will allow the construction of LIP1-deficient null mutants which will be critical in determining the role of this gene in candidal virulence.

Amino Acid Sequence↗
PubMed1997

Genes encoding the NAD-reducing hydrogenase of Rhodococcus opacus MR11.

The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus MR11 into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far. The genes of the soluble hydrogenase were localized on a 7.4 kbp Asnl fragment of the linear plasmid pHG201 via heterologous hybridization. Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hoxF, -U, -Y, -H, -W and ORF7. The six latter ORFs belong to the gene cluster of the soluble hydrogenase. Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16. The genes hoxF, -U, -Y and -H encode the subunits alpha, gamma, delta and beta, respectively. The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the beta subunit. Finally, ORF7 encodes a protein which has similarities to cAMP- and cGMP-binding protein kinases, but its function is not known. ORF1, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria. Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R. opacus MR11 indicated that the hydrogenase genes are under control of a delta 70-like promoter located at the right end of ORF1 and are even transcribed under heterotrophic conditions at a low level. Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHY1 harbouring the 7.4 kbp Asnl fragment, resulting in overexpression of the hydrogenase genes. Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E. coli, no active enzyme was detectable.

Amino Acid Sequence↗
PubMed1998

Oligopeptide permease in Borrelia burgdorferi: putative peptide-binding components encoded by both chromosomal and plasmid loci.

To elucidate the importance of oligopeptide permease for Borrelia burgdorferi, the agent of Lyme disease, a chromosomal locus in B. burgdorferi that encodes homologues of all five subunits of oligopeptide permease has been identified and characterized. B. burgdorferi has multiple copies of the gene encoding the peptide-binding component, OppA; three reside at the chromosomal locus and two are on plasmids. Northern analyses indicate that each oppA gene is independently transcribed, although the three chromosomal oppA genes are also expressed as bi- and tri-cistronic messages. Induction of one of the plasmid-encoded oppA genes was observed following an increase in temperature, which appears to be an important cue for adaptive responses in vivo. The deduced amino acid sequences suggest that all five borrelial oppA homologues are lipoproteins, but the protease-resistance of at least one of them in intact bacteria is inconsistent with outer-surface localization. Insertional inactivation of a plasmid-encoded oppA gene demonstrates that it is not essential for growth in culture.

Amino Acid Sequence↗
PubMed1999

Novel phosphotransferase system genes revealed by genome analysis - the complete complement of PTS proteins encoded within the genome of Bacillus subtilis.

Bacillus subtilis can utilize several sugars as single sources of carbon and energy. Many of these sugars are transported and concomitantly phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In addition to its role in sugar uptake, the PTS is one of the major signal transduction systems in B. subtilis. In this study, an analysis of the complete set of PTS proteins encoded within the B. subtilis genome is presented. Fifteen sugar-specific PTS permeases were found to be present and the functions of novel PTS permeases were studied based on homology to previously characterized permeases, analysis of the structure of the gene clusters in which the permease encoding genes are located and biochemical analysis of relevant mutants. Members of the glucose, sucrose, lactose, mannose and fructose/mannitol families of PTS permeases were identified. Interestingly, nine pairs of IIB and IIC domains belonging to the glucose and sucrose permease families are present in B. subtilis; by contrast only five Enzyme IIA(Glc)-like proteins or domains are encoded within the B. subtilis genome. Consequently, some of the EIIA(Glc)-like proteins must function in phosphoryl transfer to more than one IIB domain of the glucose and sucrose families. In addition, 13 PTS-associated proteins are encoded within the B. subtilis genome. These proteins include metabolic enzymes, a bifunctional protein kinase/phosphatase, a transcriptional cofactor and transcriptional regulators that are involved in PTS-dependent signal transduction. The PTS proteins and the auxiliary PTS proteins represent a highly integrated network that catalyses and simultaneously modulates carbohydrate utilization in this bacterium.

Bacillus subtilis↗
PubMed2001

The gene yghK linked to the glc operon of Escherichia coli encodes a permease for glycolate that is structurally and functionally similar to L-lactate permease.

In Escherichia coli the glc operon involved in glycolate utilization is located at 67.3 min and formed by genes encoding the enzymes glycolate oxidase (glcDEF) and malate synthase G (glcB). Their expression from a single promoter upstream of glcD is induced by growth on glycolate and regulated by the activator encoded by the divergently transcribed gene glcC. Gene yghK, located 350 bp downstream of glcB, encodes a hydrophobic protein highly similar to the L-lactate permease encoded by lldP. Expression studies have shown that the yghK gene (proposed name glcA) is transcribed from the same promoter as the other glc structural genes and thus belongs to the glc operon. Characterization of a glcA::cat mutant showed that GlcA acts as glycolate permease and that glycolate can also enter the cell through another transport system. Evidence is presented of the involvement of L-lactate permease in glycolate uptake. Growth on this compound was abolished in a double mutant of the paralogous genes glcA and lldP, and restored with plasmids expressing either GlcA or LldP. Characterization of the putative substrates for these two related permeases showed, in both cases, specificity for the 2-hydroxymonocarboxylates glycolate, L-lactate and D-lactate. Although both GlcA and LldP recognize D-lactate, mutant analysis proved that L-lactate permease is mainly responsible for its uptake.

Alcohol Oxidoreductases↗
PubMed2002

Molecular characterization and endosymbiotic localization of the gene encoding D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) form II in the deep-sea vestimentiferan trophosome.

To better understand the contribution of micro-organisms to the primary production in the deep-sea gutless tubeworm Lamellibrachia sp., the 16S-rDNA-based phylogenetic data would be complemented by knowledge of the genes that encode the enzymes relevant to chemoautotrophic carbon fixation, such as D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1.1.39). To phylogenetically characterize the autotrophic endosymbiosis within the trophosome of the tubeworm Lamellibrachia sp., bulk trophosomal DNA was extracted and analysed based on the 16S-rRNA- and RuBisCO-encoding genes. The 16S-rRNA- and RuBisCO-encoding genes were amplified by PCR, cloned and sequenced. For the 16S rDNA, a total of 50 clones were randomly selected and analysed directly by sequencing. Only one operational taxonomic unit resulted from the 16S rDNA sequence analysis. This may indicate the occurrence of one endosymbiotic bacterial species within the trophosome of the Lamellibrachia sp. used in this study. Phylogenetic analysis of the 16S rDNA showed that the Lamellibrachia sp. endosymbiont was closely related to the genus Rhodobacter, a member of the alpha-Protebacteria. For the RuBisCO genes, only the form II gene (cbbM) was amplified by PCR. A total of 50 cbbM clones were sequenced, and these were grouped into two operational RuBisCO units (ORUs) based on their deduced amino acid sequences. The cbbM ORUs showed high amino acid identities with those recorded from the ambient sediment bacteria. To confirm the results of sequence analysis, the localization of the symbiont-specific 16S rRNA and cbbM sequences in the Lamellibrachia sp. trophosome was visualized by in situ hybridization (ISH), using specific probes. Two types of cells, coccoid and filamentous, were observed at the peripheries of the trophosome lobules. Both the symbiont-specific 16S rDNA and cbbM probes hybridized at the same sites coincident with the location of the coccoid cells, whereas the filamentous cells showed no cbbM-specific signals. The RuBisCO form I gene (cbbL) was neither amplified by PCR nor detected by ISH. This is the first demonstration of chemoautotrophic symbiosis in the deep-sea gutless tubeworm, based on sequence data and in situ localization of both the 16S-rRNA- and RuBisCO-encoding genes.

Amino Acid Sequence↗