Suppressive subtractive hybridization detects extensive genomic diversity in Thermotoga maritima

Comparisons between genomes of closely related bacteria often show large variations in gene content, even between strains of the same species. Such studies have focused mainly on pathogens; here, we examined Thermotoga maritima, a free-living hyperthermophilic bacterium, by using suppressive subtractive hybridization. The genome sequence of T. maritima MSB8 is available, and DNA from this strain served as a reference to obtain strain-specific sequences from Thermotoga sp. strain RQ2, a very close relative (approximately 96% identity for orthologous protein-coding genes, 99.7% identity in the small-subunit rRNA sequence). Four hundred twenty-six RQ2 subtractive clones were sequenced. One hundred sixty-six had no DNA match in the MSB8 genome. These differential clones comprise, in sum, 48 kb of RQ2-specific DNA and match 72 genes in the GenBank database. From the number of identical clones, we estimated that RQ2 contains 350 to 400 genes not found in MSB8. Assuming a similar genome size, this corresponds to 20% of the RQ2 genome. A large proportion of the RQ2-specific genes were predicted to be involved in sugar transport and polysaccharide degradation, suggesting that polysaccharides are more important as nutrients for this strain than for MSB8. Several clones encode proteins involved in the production of surface polysaccharides. RQ2 encodes multiple subunits of a V-type ATPase, while MSB8 possesses only an F-type ATPase. Moreover, an RQ2-specific MutS homolog was found among the subtractive clones and appears to belong to a third novel archaeal type MutS lineage. Southern blot analyses showed that some of the RQ2 differential sequences are found in some other members of the order Thermotogales, but the distribution of these variable genes is patchy, suggesting frequent lateral gene transfer within the group.     

Genome sequence of Thermotoga sp. strain RQ2, a hyperthermophilic bacterium isolated from a geothermally heated region of the seafloor near Ribeira Quente, the Azores

Thermotoga sp. strain RQ2 is probably a strain of Thermotoga maritima. Its complete genome sequence allows for an examination of the extent and consequences of gene flow within Thermotoga species and strains. Thermotoga sp. RQ2 differs from T. maritima in its genes involved in myo-inositol metabolism. Its genome also encodes an apparent fructose phosphotransferase system (PTS) sugar transporter. This operon is also found in Thermotoga naphthophila strain RKU-10 but no other Thermotogales. These are the first reported PTS transporters in the Thermotogales.     

Phylogenetic analyses of two "archaeal" genes in thermotoga maritima reveal multiple transfers between archaea and bacteria

The genome sequence of Thermotoga maritima revealed that 24% of its open reading frames (ORFs) showed the highest similarity scores to archaeal genes in BLAST analyses. Here we screened 16 strains from the genus Thermotoga and other related Thermotogales for the occurrence of two of these "archaeal" genes: the gene encoding the large subunit of glutamate synthase (gltB) and the myo-inositol 1P synthase gene (ino1). Both genes were restricted to the Thermotoga species within the Thermotogales. The distribution of the two genes, along with results from phylogenetic analyses, showed that they were acquired from Archaea during the divergence of the Thermotogales. Database searches revealed that three other bacteria-Dehalococcoides ethenogenes, Sinorhizobium meliloti, and Clostridium difficile-possess archaeal-type gltBs, and the phylogenetic analyses confirmed at least two lateral gene transfer (LGT) events between Bacteria and Archaea. These LGT events were also strongly supported by gene structure data, as the three domains in bacterial-type gltB are homologous to three independent ORFs in Archaea and Bacteria with archaeal-type gltBs. The ino1 gene has a scattered distribution among Bacteria, and apart from the Thermotoga strains it is found only in Aquifex aeolicus, D. ethenogenes, and some high-G+C Gram-positive bacteria. Phylogenetic analysis of the ino1 sequences revealed three highly supported prokaryotic clades, all containing a mixture of archaeal and bacterial sequences, and suggested that all bacterial ino1 genes had been recruited from archaeal donors. The Thermotoga strains and A. aeolicus acquired this gene independently from different archaeal species. Although transfer of genes from hyperthermophilic Archaea may have facilitated the evolution of bacterial hyperthermophily, between-domain transfers also affect mesophilic species. For hyperthermophiles, we hypothesize that LGT may be as much a consequence as the cause of adaptation to hyperthermophily.     

Cloning and sequencing of the genes of 2-hydoxyglutaryl-CoA dehydratase from Acidaminococcus fermentans

Two genomic libraries from Acidaminococcus fermentans DNA constructed with the lambda vectors gt11 and EMBL 3 were screened with antisera raised against 2-hydroxyglutaryl-CoA dehydratase. Two clones giving the strongest reaction in the immunoassay were analyzed further, one was a lambda gt11 clone with an insert of 2050 bp and one was a lambda EMBL-3 clone with an insert of approximately 11,000 bp. Escherichia coli cells infected with the lambda gt11 clone expressed the alpha subunit of the dehydratase (Mr, 53,870), whereas with the lambda EMBL-3 clone, the alpha and beta subunits (Mr, 41,857) were detected on Western blots. Restriction fragments of both clones were subcloned in pUC 8 and sequenced by the chain termination method. Thus the complete sequence of the genes of both subunits, hgdA (alpha) and hgdB (beta) were obtained. The genes have the following order: A-B, with an intergenic region of only 2 bp. The deduced amino acid sequences for the alpha and beta subunits were confirmed by four peptides sequenced by protein chemical methods. Both chains are extremely rich in cysteine (13 in alpha, including a CNC and two CC clusters, and nine in beta) but no similarities to other known protein sequences were found.     

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt

Abstract We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that the analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema . It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.     

Preparation of DNA libraries of Plasmodium falciparum for searching calmodulin binding protein genes,GOGAT and Pfmyo A

A cDNA library of Plasmodium falciparum (Colombian strain FCB2) asexual stage was constructed in the lambda ZipLox vector. The lambda ZipLox library and a lambda ZAPII (Dd2 strain) were screened for genes coding for proteins that bind with or are related to calmodulin (CaM). Screening was accomplished with Hot start PCR assays and hybridization with radiolabeled probes. Actin I, CaM, glutamate synthase (GOGAT) and the three myosin clones--Pfmyo A, Pfmyo B and Pfmyo C--were identified. The clones coding for actin I, CaM and GOGAT were retrieved from the lambda ZipLox library, and the GOGAT and Pfmyo A clones from the lambda ZAP II library. The GOGAT clone contained an insert of 2,413 base pairs corresponding to 24.8% of the reported sequence. The Pfmyo A insert was 2,457 base pairs long, and represented the complete mRNA coding for this gene. Finally, the first report of a complete cDNA clone containing the P. falciparum myosin A is presented.     

Phylogenetic Diversity of Archaea and Bacteria in a Deep Subsurface Paleosol

Abstract A low-biomass paleosol 188 m below the ground surface at the Department of Energy's Hanford Site in south-central Washington State was recovered and maintained at the in situ temperature (17°C) as an intact core or homogenized sediment for 0, 1, 3, 10, and 21 weeks post-sampling. Bacterial and archaeal 16S rRNA genes were amplified by PCR and cloned. Of 746 bacterial and 190 archaeal clones that were categorized by restriction fragment length polymorphism (RFLP), 242 bacterial and 16 archaeal clones were partially sequenced and compared against the small subunit ribosomal RNA database (RDP) and GenBank. Six bacterial and 16 archaeal clones sequences, with little similarity to those in public databases, were sequenced in their entirety, and subjected to more detained phylogenetic analysis. The most frequently occurring clones types were related to Pseudomonas, Bacillus, Micrococcus, Clavibacter, Nocardioides, Burkholderia, Comamonas, and Erythromicrobium. Clone sequences whose RDP similarity value was ≥0.6 consistently grouped with their nearest RDP neighbor during phylogenetic analysis. Six truly novel eubacterial sequences were identified; they consistently cluster with or near the Chloroflexaceae and sequences recovered from the Sargasso Sea. Sixteen unique archaeal RFLP groups were identified from 190 randomly-sampled clones. The novel archaeal rDNA clones formed a coherent clade along the major Crenarchaea branch containing all previously described mesophilic crenarchae clones, but remained firmly associated with 16S rDNA clones previously obtained from a thermal Fe/S spring in Yellowstone National Park. The wealth of group-specific genetic information identified during this study will now allow us to address specific hypotheses related to in situ stimulation of these deep subsurface microorganisms and changes in microbial community composition resulting from subsurface contamination or remediation processes at the Hanford Site. Revised: 21 October 1997; Accepted: 20 November 1997     

Soluble methane monooxygenase gene clusters from trichloroethylene-degrading Methylomonas sp. strains and detection of methanotrophs during in situ bioremediation

The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb KpnI fragment from Methylomonas sp. strain KSWIII and a 7.5-kb SalI fragment from Methylomonas sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from M. capsulatus Bath in the group X methanotrophs than to those from Methylosinus trichosporium OB3b and Methylocystis sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the mmoX gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.     

Identification of an enzyme system for daidzein-to-equol conversion in Slackia sp. strain NATTS

An Escherichia coli library comprising 8,424 strains incorporating gene fragments of the equol-producing bacterium Slackia sp. strain NATTS was constructed and screened for E. coli strains having daidzein- and dihydrodaidzein (DHD)- metabolizing activity. We obtained 3 clones that functioned to convert daidzein to DHD and 2 clones that converted DHD to equol. We then sequenced the gene fragments inserted into plasmids contained by these 5 clones. All of the gene fragments were contiguous, encoding three open reading frames (ORF-1, -2, and -3). Analysis of E. coli strains containing an expression vector incorporating one of the orf-1, -2, or -3 genes revealed that (i) the protein encoded by orf-1 was involved in the conversion of cis/trans-tetrahydrodaidzein (cis/trans-THD) to equol, (ii) the protein encoded by orf-2 was involved in the conversion of DHD to cis/trans-THD, and (iii) the protein encoded by orf-3 was involved in the conversion of daidzein to DHD. ORF-1 had a primary amino acid structure similar to that of succinate dehydrogenase. ORF-2 was presumed to be an enzyme belonging to the short-chain dehydrogenase/reductase superfamily. ORF-3 was predicted to have 42% identity to the daidzein reductase of Lactococcus strain 20-92 and belonged to the NADH:flavin oxidoreductase family. These findings showed that the daidzein-to-equol conversion reaction in the Slackia sp. NATTS strain proceeds by the action of these three enzymes.     

cDNA clones encoding rabbit immunoglobulin lambda chains. Evidence for length variation of the third hypervariable region and for a novel constant region.

Five cDNA clones designated pDH2, pDH8, pDH9, pDH31 and pDH101 encoding rabbit immunoglobulin lambda light chain sequences have been characterized. Comparison of the V lambda sequences suggests that, in addition to an increased divergence in all of the complementarity-determining regions (CDRs), variable-region diversity is amplified by the length heterogeneity of the CDR3, at the V lambda-J lambda junction. An insertion of four codons at positions 48a-d has been noted in three cDNA sequences. This insert, not found in lambda nor kappa light chains of other species, has a variable sequence, suggesting its possible implication in expanding variability of the CDR2. One of the cDNA clones was shown to encode a novel C lambda region which differs by four amino acid substitutions from the C lambda region common to all the other clones. Thus, the rabbit can use two different C lambda genes, which might correlate with the expression of the two known allotypes of lambda chains, C7 and C21. Southern blotting experiments indicate a small number of germ-line V lambda genes and the cDNA nucleotide sequence data reported here suggest that several of these genes can be expressed. The possibility of at least two V-J-C gene clusters is discussed.     

Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis.

The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers IAf and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the alpha-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed marked variation from the remainder in specific regions of the sequence. From a comparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe was used to screen primary populations of PCR clones, and all of those that were probe negative were checked for the presence of inserts, which were then sequenced. By using this strategy, further novel methanogen 16S rRNA variants were identified and analyzed. The sequences recovered from the peat formed two clusters on the end of long branches within the methanogen radiation that are distinct from each other. These cannot be placed directly with sequences from any cultured taxa for which sequence information is available.     

Isolation and Characterization of ACC Deaminase Gene from Two Plant Growth-Promoting Rhizobacteria

Lowering of plant ethylene by deamination of its immediate precursor 1-aminocyclopropane-1-carboxylate (ACC) is a key trait found in many rhizobacteria. We isolated and screened bacteria from the rhizosphere of wheat for their ACC-degrading ability. The ACC deaminase gene (acdS) isolated from two bacterial isolates through PCR amplification was cloned and sequenced. Nucleotide sequence alignment of these genes with previously reported genes of Pseudomonas sp. strain ACP and Enterobacter cloacae strain UW4 showed variation in their sequences. In the phylogenetic analysis, distinctness of these two genes was observed as a separate cluster. 16S rDNA sequencing of two isolates identified them to be Achromobacter sp. and Pseudomonas stutzeri.     

Archaeal diversity in waters from deep South African gold mines.

A culture-independent molecular analysis of archaeal communities in waters collected from deep South African gold mines was performed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rRNA genes (rDNA) in conjunction with a sequencing analysis of archaeal rDNA clone libraries. The water samples used represented various environments, including deep fissure water, mine service water, and water from an overlying dolomite aquifer. T-RFLP analysis revealed that the ribotype distribution of archaea varied with the source of water. The archaeal communities in the deep gold mine environments exhibited great phylogenetic diversity; the majority of the members were most closely related to uncultivated species. Some archaeal rDNA clones obtained from mine service water and dolomite aquifer water samples were most closely related to environmental rDNA clones from surface soil (soil clones) and marine environments (marine group I [MGI]). Other clones exhibited intermediate phylogenetic affiliation between soil clones and MGI in the Crenarchaeota. Fissure water samples, derived from active or dormant geothermal environments, yielded archaeal sequences that exhibited novel phylogeny, including a novel lineage of Euryarchaeota. These results suggest that deep South African gold mines harbor novel archaeal communities distinct from those observed in other environments. Based on the phylogenetic analysis of archaeal strains and rDNA clones, including the newly discovered archaeal rDNA clones, the evolutionary relationship and the phylogenetic organization of the domain Archaea are reevaluated.     

Archaeal diversity in tidal flat sediment as revealed by 16S rDNA analysis

During the past ten years, Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages. More recently, the presence of novel archaeal phylogenetic lineages has been discovered in coastal marine environments, freshwater lakes, polar seas, and deep-sea hydrothermal vents. Therefore, we conducted an investigation into the archaeal community existing in tidal flat sediment collected from Ganghwa Island, Korea. Phylogenetic analysis of archaeal 16S rDNA amplified directly from tidal flat sediment DNA revealed the presence of two major lineages, belonging to the Crenarchaeota (53.9%) and Euryarchaeota (46.1%) phyla. A total of 102 clones were then sequenced and analyzed by comprehensive phylogenetic analysis. The sequences determined in our samples were found to be closely related to the sequences of clones which had been previously obtained from a variety of marine environments. Archaeal clones exhibited higher similarities (83.25-100%) to sequences from other environments in the public database than did those (75.22-98.46%) of previously reported bacterial clones obtained from tidal flat sediment. The results of our study suggest that the archaeal community in tidal flat sediment is remarkably diverse.     

Ordered shotgun sequencing of a 135 kb Xq25 YAC containing ANT2 and four possible genes, including three confirmed by EST matches.

Ordered shotgun sequencing (OSS) has been successfully carried out with an Xq25 YAC substrate. yWXD703 DNA was subcloned into lambda phage and sequences of insert ends of the lambda subclones were used to generate a map to select a minimum tiling path of clones to be completely sequenced. The sequence of 135 038 nt contains the entire ANT2 cDNA as well as four other candidates suggested by computer-assisted analyses. One of the putative genes is homologous to a gene implicated in Graves' disease and it, ANT2 and two others are confirmed by EST matches. The results suggest that OSS can be applied to YACs in accord with earlier simulations and further indicate that the sequence of the YAC accurately reflects the sequence of uncloned human DNA.     

Archaeal Diversity in Waters from Deep South African Gold Mines

A culture-independent molecular analysis of archaeal communities in waters collected from deep South African gold mines was performed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rRNA genes (rDNA) in conjunction with a sequencing analysis of archaeal rDNA clone libraries. The water samples used represented various environments, including deep fissure water, mine service water, and water from an overlying dolomite aquifer. T-RFLP analysis revealed that the ribotype distribution of archaea varied with the source of water. The archaeal communities in the deep gold mine environments exhibited great phylogenetic diversity; the majority of the members were most closely related to uncultivated species. Some archaeal rDNA clones obtained from mine service water and dolomite aquifer water samples were most closely related to environmental rDNA clones from surface soil (soil clones) and marine environments (marine group I [MGI]). Other clones exhibited intermediate phylogenetic affiliation between soil clones and MGI in the Crenarchaeota. Fissure water samples, derived from active or dormant geothermal environments, yielded archaeal sequences that exhibited novel phylogeny, including a novel lineage of Euryarchaeota. These results suggest that deep South African gold mines harbor novel archaeal communities distinct from those observed in other environments. Based on the phylogenetic analysis of archaeal strains and rDNA clones, including the newly discovered archaeal rDNA clones, the evolutionary relationship and the phylogenetic organization of the domain Archaea are reevaluated.     

Diversity of radA Genes from Cultured and Uncultured Archaea: Comparative Analysis of Putative RadA Proteins and Their Use as a Phylogenetic Marker

Archaea-specific radA primers were used with PCR to amplify fragments of radA genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15 radA genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species of Crenarcheota. The two most deeply branching archaeal radA genes found had some amino acid deletion and insertion patterns characteristic of bacterial recA genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.     

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.     

Construction of a bacterial artificial chromosome (BAC) library of Lycopersicon esculentum cv. Stevens and its application to physically map the Sw-5 locus

The Sw-5 gene is a dominantly inherited resistance gene in tomato and functional against a number of tospovirus species. The gene has been mapped on chromosome 9, tightly linked to RFLP markers CT220 and SCAR421. To analyse the Sw-5 locus, a BAC genomic library was constructed of tomato cv. Stevens, homozygous for the Sw-5 gene. The library comprised 18 816 clones with an average insert size of 100 kb, corresponding to two genome equivalents. The library was screened by PCR using primers designed for the CT220 and SCAR421 sequences, resulting in a 250 kb contig of known orientation on the long arm of chromosome 9. Using degenerate primers based on homologous sequences in the nucleotide binding site of resistance gene sequences, three discrete PCR fragments obtained from this contig were cloned and sequenced. Analysis of these fragments revealed a high similarity with numerous resistance genes or resistance gene like sequences. The present data indicate that at least three different resistance gene candidate (RGC) sequences are present in the vicinity of marker CT220, supporting the view that a resistance gene family may be responsible for the unusually broad resistance to tospoviruses conferred by the Sw-5 locus. This revised version was published online in June 2006 with corrections to the Cover Date.