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.     

Targeting clusters of transferred genes in Thermotoga maritima

We screened a Thermotoga sp. strain RQ2 lambda library for genes present in that strain but absent from the closely related completely sequenced relative Thermotoga maritima strain MSB8, by using probes generated in an earlier genomic subtraction study. Five lambda insert fragments were sequenced, containing, respectively, an archaeal type ATPase operon, rhamnose biosynthetic genes, ORFs with similarity to an arabinosidase, a Thermotoga sp. strain RQ2-specific alcohol dehydrogenase and a novel archaeal Mut-S homologue. All but one of these fragments contained additional Thermotoga sp. strain RQ2-specific sequences not screened for, suggesting that many such strain-specific genes will be found clustered in the genome. Moreover, phylogenetic analyses, phylogenetic distribution and/or G + C content suggests that all the Thermotoga sp. strain RQ2 specific sequences in the sequenced lambda clones have been acquired by lateral gene transfer. We suggest that the use of strain-specific small insert clones obtained by subtractive hybridization to target larger inserts for sequencing is an efficient, economical way to identify environmentally (or clinically) relevant interstrain differences and novel gene clusters, and will be invaluable in comparative genomics.     

极端耐热木聚糖酶基因在大肠杆菌和毕赤酵母中的高效表达

以海栖热袍菌 (Thermotoga maritima) MSB8菌株基因组DNA为模板,通过PCR扩增出木聚糖酶（XylanaseB）基因, 将此基因克隆至大肠杆菌表达载体pET_28a（+）和毕赤酵母表达载体pPIC9K,并分别转化大肠杆菌 BL21和毕赤酵母GS115。该木聚糖酶在大肠杆菌细胞中表达量高, 但不能分泌; 而在毕赤酵母细胞的表达产物可分泌至胞外。酶学性质分析表明,此酶分子量约为40kD,其最适反应温度为90℃, 最适反应pH值为6.65,且在碱性条件下稳定,具有重要的工业应用前景。     

Local variability of the phosphoglycerate kinase-triosephosphate isomerase fusion protein from Thermotoga maritima MSB 8

The pgk-tpi gene locus of Thermotoga maritima encodes both phosphoglycerate kinase (PGK) and a bienzyme complex consisting of a fusion protein of PGK with triosephosphate isomerase (TIM). No separate tpi gene for TIM is present in T. maritima. A frame-shift at the end of the pgk gene has been previously proposed as a mechanism to regulate the expression of the two protein variants [Schurig et al., EMBO J. 14 (1995), 442-451]. Surprisingly, the complete T. maritima genome was found to contain a pgk-tpi sequence not requiring the proposed frameshift mechanism. To clarify the apparent discrepancy, a variety of DNA sequencing techniques were applied, disclosing an anomalous local variability in the pgk-tpi fusion region. The comparison of different DNA samples and the mass spectrometric analysis of the amino acid sequence of the natural fusion protein from T. maritima MSB8 confirmed the local variability of the DNA variants. Since not all peptide masses could be assigned, further variations are conceivable, suggesting an even higher heterogeneity of the T. maritima MSB8 strain.     

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 characterization of β-galactoside and β-glucoside hydrolysing enzymes of Thermotoga maritima

Abstract A gene library of the hyperthermophilic bacterium Thermotoga maritima strain MSB8 was constructed in Escherichia coli . Two non-related T. maritima chromosomal DNA fragments were physically characterized. They conferred the synthesis of thermostable X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside)-hydrolysing activity upon the host organism. The biochemical properties of the recombinant enzymes indicated that genes for a β-galactosidase (BgaA) and a broad-specificity β-glucosidase (Bg1A) had been isolated. The genes were desiignted bgaA and bglA , respectively. According to analytical size exclusion chromatography data, BgaA and BglA had native molecular masses of approximately 240 kDa and 95 kDa, respectively. Both enzymes apparently have dimeric subunit structure. An additional β-glucosidase (designated BglB) activity, clearly distinct from BglA in terms of substrate specificity, could be detected in a crude extract of T. maritima .     

F- and V-type ATPases in the hyperthermophilic bacterium Thermotoga neapolitana

Two gene clusters encoding F- or V-type ATPases were found in genomic DNA of the hyperthermophilic bacterium Thermotoga neapolitana. The subunit genes of each ATPase formed an operon. While the gene arrangement in the operon of the F-type ATPase resembled those in eukaryotic organelles and bacteria, that of the V-type ATPase was different from those reported for archaea, bacteria, or eukaryotes. Both ATPases were found to be expressed in the cells of T. neapolitana by Western blot analysis. Although V-type ATPase could not be rendered soluble, F-type ATPase was solubilized with 1% Triton X-100 and characterized. This is the first report of the coexistence of both F- and V-type ATPases in hyperthermophilic bacteria. It has recently been shown by a genome analysis that Thermotoga maritima has no V-type ATPase gene cluster but does have an F-type ATPase gene cluster; however, part of a gene for the D-subunit of the V-type ATPase gene has been reported in the T. maritima genome. Evolution of the two types of ATPases in Thermotoga is discussed.     

Thermotoga heats up lateral gene transfer.

The complete sequence of the bacterium Thermotoga maritima genome has revealed a large fraction of genes most closely related to those of archaeal species. This adds to the accumulating evidence that lateral gene transfer is a potent evolutionary force in prokaryotes, though questions of its magnitude remain.     

Chromosome evolution in the Thermotogales: large-scale inversions and strain diversification of CRISPR sequences

In the present study, the chromosomes of two members of the Thermotogales were compared. A whole-genome alignment of Thermotoga maritima MSB8 and Thermotoga neapolitana NS-E has revealed numerous large-scale DNA rearrangements, most of which are associated with CRISPR DNA repeats and/or tRNA genes. These DNA rearrangements do not include the putative origin of DNA replication but move within the same replichore, i.e., the same replicating half of the chromosome (delimited by the replication origin and terminus). Based on cumulative GC skew analysis, both the T. maritima and T. neapolitana lineages contain one or two major inverted DNA segments. Also, based on PCR amplification and sequence analysis of the DNA joints that are associated with the major rearrangements, the overall chromosome architecture was found to be conserved at most DNA joints for other strains of T. neapolitana. Taken together, the results from this analysis suggest that the observed chromosomal rearrangements in the Thermotogales likely occurred by successive inversions after their divergence from a common ancestor and before strain diversification. Finally, sequence analysis shows that size polymorphisms in the DNA joints associated with CRISPRs can be explained by expansion and possibly contraction of the DNA repeat and spacer unit, providing a tool for discerning the relatedness of strains from different geographic locations.     

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.     

海栖热袍菌葡萄糖异构酶基因xylA的克隆、表达及酶学性质

海栖热袍菌(Thermotoga maritima)是嗜极端高温的厌氧细菌,其产生的葡萄糖异构酶由于其出色的耐热性有着潜在的工业应用价值.由于海栖热袍菌苛刻的培养条件导致其葡萄糖异构酶产量较低.通过PCR方法克隆编码T. maritima MSB8葡萄糖异构酶基因xylA,构建重组质粒pHsh-xylA,转入Escherichia coli JM109,通过热激诱导表达.通过热处理和离子交换层析纯化两步得到电泳纯的酶制品,纯化倍数和回收率分别为8.02和49.02.对酶学性质研究表明,该重组酶为金属离子激活性酶,Mg2 ,Co2 对相对酶活有很强的激活作用,其最适pH为7.0,最适反应温度为95℃,且在pH 6～8之间有着较好的稳定性,在95℃下半衰期长达5 h以上.以葡萄糖为底物时的表观Km和Vmax分别为105 mmol/L和45.2 mol/min·mg.     

Properties and gene structure of the Thermotoga maritima alpha-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium.

Thermotoga maritima MSB8 has a chromosomal alpha-amylase gene, designated amyA, that is predicted to code for a 553-amino-acid preprotein with significant amino acid sequence similarity to the 4-alpha-glucanotransferase of the same strain and to alpha-amylase primary structures of other organisms. Upstream of the amylase gene, a divergently oriented open reading frame which can be translated into a polypeptide with similarity to the maltose-binding protein MalE of Escherichia coli was found. The T. maritima alpha-amylase appears to be the first known example of a lipoprotein alpha-amylase. This is in agreement with observations pointing to the membrane localization of this enzyme in T. maritima. Following the signal peptide, a 25-residue putative linker sequence rich in serine and threonine was found. The amylase gene was expressed in E. coli, and the recombinant enzyme was purified and characterized. The molecular mass of the recombinant enzyme was estimated at 61 kDa by denaturing gel electrophoresis (63 kDa by gel permeation chromatography). In a 10-min assay at the optimum pH of 7.0, the optimum temperature of amylase activity was 85 to 90 degrees C. Like the alpha-amylases of many other organisms, the activity of the T. maritima alpha-amylase was dependent on Ca2+. The final products of hydrolysis of soluble starch and amylose were mainly glucose and maltose. The extraordinarily high specific activity of the T. maritima alpha-amylase (about 5.6 x 10(3) U/mg of protein at 80 degrees C, pH 7, with amylose as the substrate) together with its extreme thermal stability makes this enzyme an interesting candidate for biotechnological applications in the starch processing industry.     

A scaleable and integrated crystallization pipeline applied to mining the Thermotoga maritima proteome

The wealth of genomic data available for many organisms has set the stage for the next phase of structure-function analysis. High-throughput structural genomics is currently the method of choice for rapid analysis of protein structure-function relationships on a proteome-wide basis. The Joint Center for Structural Genomics (JCSG), established in 2000 under the NIH/NIGMS Protein Structure Initiative, has developed and implemented an integrated high-throughput structure pipeline and applied it in a 2-tiered approach to mining the proteome of the thermophilic bacterium Thermotoga maritima. In the first tier, the successful application of this integrated pipeline has resulted in the cloning and expression of 73% of the T. maritima proteome (1376 out of 1877 predicted genes), and has identified 465 proteins which produced crystal hits. These 465 proteins were compared with existing structural information and a subset of 269 targets were selected to process towards structure determination in a second tier effort. To date, the JCSG pipeline applied to the Thermotoga maritima proteome has resulted in 55 new structures and has identified 6 novel folds and continues to identify structures with novel features.     

An oxygen reduction chain in the hyperthermophilic anaerobe Thermotoga maritima highlights horizontal gene transfer between Thermococcales and Thermotogales

The hyperthermophile Thermotoga maritima, although strictly anaerobic, is able to grow in the presence of low amounts of O(2). Here, we show that this bacterium consumes O(2) via a three-partner chain involving an NADH oxidoreductase (NRO), a rubredoxin (Rd) and a flavo-diiron protein (FprA) (locus tags: TM_0754, TM_0659 and TM_0755, respectively). In vitro experiments showed that the NADH-dependent O(2) consumption rate was 881.9 (± 106.7) mol O(2) consumed min(-1) per mol of FprA at 37°C and that water was the main end-product of the reaction. We propose that this O(2) reduction chain plays a central role in the O(2) tolerance of T. maritima. Phylogenetic analyses suggest that the genes coding for these three components were acquired by an ancestor of Thermotogales from an ancestor of Thermococcales via a single gene transfer. This event likely also involved two ROS scavenging enzymes (neelaredoxin and rubrerythrin) that are encoded by genes clustered with those coding for FprA, NRO and Rd in the ancestor of Thermococcales. Such genomic organization would have provided the ancestor of Thermotogales with a complete set of enzymes dedicated to O(2)-toxicity defence. Beside Thermotogales and Thermococcales, horizontal gene transfers have played a major role in disseminating these enzymes within the hyperthermophilic anaerobic prokaryotic communities, allowing them to cope with fluctuating oxidative conditions that exist in situ.