The genome of AR9, a giant transducing Bacillus phage encoding two multisubunit RNA polymerases.

Bacteriophage AR9 and its close relative PBS1 have been extensively used to construct early Bacillus subtilis genetic maps. Here, we present the 251,042bp AR9 genome, a linear, terminally redundant double-stranded DNA containing deoxyuridine instead of thymine. Multiple AR9 genes are interrupted by non-coding sequences or sequences encoding putative endonucleases. We show that these sequences are group I and group II self-splicing introns. Eight AR9 proteins are homologous to fragments of bacterial RNA polymerase (RNAP) subunits ß/ß'. These proteins comprise two sets of paralogs of RNAP largest subunits, with each paralog encoded by two disjoint phage genes. Thus, AR9 is a phiKZ-related giant phage that relies on two multisubunit viral RNAPs to transcribe its genome independently of host transcription apparatus. Purification of one of PBS1/AR9 RNAPs has been reported previously, which makes AR9 a promising object for further studies of RNAP evolution, assembly and mechanism.

Draft Genome Sequence of the Fungus Trametes hirsuta 072.

A standard draft genome sequence of the white rot saprotrophic fungus Trametes hirsuta 072 (Basidiomycota, Polyporales) is presented. The genome sequence contains about 33.6 Mb assembled in 141 scaffolds with a G+C content of ~57.6%. The draft genome annotation predicts 14,598 putative protein-coding open reading frames (ORFs).

Complete Genome Sequence of Lactobacillus acidophilus FSI4, Isolated from Yogurt.

A new Lactobacillus acidophilus strain, FSI4, isolated from yogurt, was isolated and sequenced in our laboratory. Our data, although supportive of previous conclusions regarding the remarkable stability of L. acidophilus species, indicate accumulating mutations in commercial L. acidophilus strains that warrant further study of the effect of damaged genes on the competitiveness of these bacteria in gut microbiota.

Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

Genome comparison and proteomic characterization of Thermus thermophilus bacteriophages P23-45 and P74-26: siphoviruses with triplex-forming sequences and the longest known tails.

The genomes of two closely related lytic Thermus thermophilus siphoviruses with exceptionally long (approximately 800 nm) tails, bacteriophages P23-45 and P74-26, were sequenced completely. The P23-45 genome consists of 84,201 bp with 117 putative open reading frames (ORFs), and the P74-26 genome has 83,319 bp and 116 putative ORFs. The two genomes are 92% identical with 113 ORFs shared. Only 25% of phage gene product functions can be predicted from similarities to proteins and protein domains with known functions. The structural genes of P23-45, most of which have no similarity to sequences from public databases, were identified by mass spectrometric analysis of virions. An unusual feature of the P23-45 and P74-26 genomes is the presence, in their largest intergenic regions, of long polypurine-polypyrimidine (R-Y) sequences with mirror repeat symmetry. Such sequences, abundant in eukaryotic genomes but rare in prokaryotes, are known to form stable triple helices that block replication and transcription and induce genetic instability. Comparative analysis of the two phage genomes shows that the area around the triplex-forming elements is enriched in mutational variations. In vitro, phage R-Y sequences form triplexes and block DNA synthesis by Taq DNA polymerase in orientation-dependent manner, suggesting that they may play a regulatory role during P23-45 and P74-26 development.

Thermus thermophilus bacteriophage phiYS40 genome and proteomic characterization of virions.

We determined the sequence of the 152,372 bp genome of phiYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of phiYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. phiYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of phiYS40, most of which have no similarity to sequences in public databases, were identified by mass spectrometric analysis of purified virions. Various phiYS40 proteins have different phylogenetic neighbors, including myovirus, podovirus, and siphovirus gene products, bacterial genes and, in one case, a dUTPase from a eukaryotic virus. phiYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.

High-throughput production of optimized primers (fimers) for whole-genome direct sequencing.

Fimers are specifically modified primers selected to inhibit nonspecific interactions occurring in cycle sequencing. They are postsynthetically derived from 2'-methoxyoxalamido or 2'-succinimido precursor oligonucleotides by treatment with appropriate small molecular weight modifiers (a primary aliphatic amine or hydroxide anion). We describe design, synthesis, and purification of fimers, and their use in protocols for direct sequencing of genomic deoxyribonucleic acid (DNA). Protocol for isolation of microbial genomic DNA is also reported.

Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases.

Helix-hairpin-helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)(2) domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH(2) terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1- and polB-type polymerases, as well as to other DNA processing enzymes.

The complete genome of hyperthermophile Methanopyrus kandleri AV19 and monophyly of archaeal methanogens.

We have determined the complete 1,694,969-nt sequence of the GC-rich genome of Methanopyrus kandleri by using a whole direct genome sequencing approach. This approach is based on unlinking of genomic DNA with the ThermoFidelase version of M. kandleri topoisomerase V and cycle sequencing directed by 2'-modified oligonucleotides (Fimers). Sequencing redundancy (3.3x) was sufficient to assemble the genome with less than one error per 40 kb. Using a combination of sequence database searches and coding potential prediction, 1,692 protein-coding genes and 39 genes for structural RNAs were identified. M. kandleri proteins show an unusually high content of negatively charged amino acids, which might be an adaptation to the high intracellular salinity. Previous phylogenetic analysis of 16S RNA suggested that M. kandleri belonged to a very deep branch, close to the root of the archaeal tree. However, genome comparisons indicate that, in both trees constructed using concatenated alignments of ribosomal proteins and trees based on gene content, M. kandleri consistently groups with other archaeal methanogens. M. kandleri shares the set of genes implicated in methanogenesis and, in part, its operon organization with Methanococcus jannaschii and Methanothermobacter thermoautotrophicum. These findings indicate that archaeal methanogens are monophyletic. A distinctive feature of M. kandleri is the paucity of proteins involved in signaling and regulation of gene expression. Also, M. kandleri appears to have fewer genes acquired via lateral transfer than other archaea. These features might reflect the extreme habitat of this organism.