Project description:Although mitochondria and chloroplasts are considered to be descendants of eubacteria-like endo- symbionts, the mitochondrial RNA polymerase of yeast is a nucleus-encoded, single-subunit enzyme homologous to bacteriophage T3 and T7 RNA polymerases, rather than a multi-component, eubacterial-type alpha 2 beta beta' enzyme, as encoded in chloroplast DNA. To broaden our knowledge of the mitochondrial transcriptional apparatus, we have used a polymerase chain reaction (PCR) approach designed to amplify an internal portion of phage T3/T7-like RNA polymerase genes. Using this strategy, we have recovered sequences homologous to yeast mitochondrial and phage T3/T7 RNA polymerases from a phylogenetically broad range of multicellular and unicellular eukaryotes. These organisms display diverse patterns of mitochondrial genome organization and expression, and include species that separated from the main eukaryotic line early in the evolution of this lineage. In certain cases, we can deduce that PCR-amplified sequences, some of which contain small introns, are localized in nuclear DNA. We infer that the T3/T7-like RNA polymerase sequences reported here are likely derived from genes encoding the mitochondrial RNA polymerase in the organisms in which they occur, suggesting a phage T3/T7-like RNA polymerase was recruited to act in transcription in the mitochondrion at an early stage in the evolution of this organelle.
Project description:In most organisms, the mitochondrial genes are transcribed by RNA polymerases related to the single-subunit RNA polymerases of bacteriophages like T3 and T7. In flowering plants, duplication(s) of the RpoTm gene coding for the mitochondrial RNA polymerase (RPOTm) led to the evolution of additional RNA polymerases transcribing genes in plastids (RPOTp) or in both mitochondria and plastids (RPOTmp). Two putative RPOTmp enzymes were previously described to be encoded by the nuclear genes RpoTmp1 and RpoTmp2 in the moss Physcomitrella patens. Here, we report on a third Physcomitrella RpoT gene. We determined the sequence of the cDNA. Comparison of the deduced amino acid sequence with sequences of plant organellar RNA polymerases suggests that this gene encodes a functional phage-type RNA polymerase. The 78 N-terminal amino acids of the putative RNA polymerase were fused to GFP and found to target the fusion protein exclusively to mitochondria in Arabidopsis protoplasts. P. patens is the only known organism to possess three mitochondrial RNA polymerases.
Project description:It is well appreciated that the evolutionary divergence of genes and genomes from a common ancestor ultimately leads to incompatibilities if those genomes are hybridized. Far less is known about the ability and nature of compensatory evolution to yield the recovery of function in hybrid genomes. Here the major capsid gene of the bacteriophage T7 (40-kb dsDNA) was replaced with the homologous gene of either T3 or K11, each 22% different at the protein level from the T7 homolog. Initial fitness was moderately impaired for the T3 exchange, but the K11 exchange was not viable without a compensatory change in the T7 scaffolding protein. Subsequent adaptation of the transgenic phages led to nearly complete fitness recoveries. Compensatory changes were few, mostly in the transgene and its main interacting partner, the scaffolding protein gene. The large magnitude of fitness recovery with relatively few mutations suggests that the fitness costs of hybridizations and horizontal gene exchanges between moderately diverged genomes can potentially be short-lived through compensatory evolution.
Project description:It is usually thought that bacteriophage T7 is female specific, while phage T3 can propagate on male and female Escherichia coli. We found that the growth patterns of phages T7M and T3 do not match the above characteristics, instead showing strain dependent male exclusion. Furthermore, a T3/7 hybrid phage exhibits a broader host range relative to that of T3, T7, as well as T7M, and is able to overcome the male exclusion. The T7M sequence closely resembles that of T3. T3/7 is essentially T3 based, but a DNA fragment containing part of the tail fiber gene 17 is replaced by the T7 sequence. T3 displays inferior adsorption to strains tested herein compared to T7. The T3 and T7 recombinant phage carries altered tail fibers and acquires better adsorption efficiency than T3. How phages T3 and T7 recombine was previously unclear. This study is the first to show that recombination can occur accurately within only 8 base-pair homology, where four-way junction structures are identified. Genomic recombination models based on endonuclease I cleavages at equivalent and nonequivalent sites followed by strand annealing are proposed. Retention of pseudo-palindromes can increase recombination frequency for reviving under stress.
Project description:Due to antimicrobial resistance and the public health hazard of antibiotic growth promoters, there is a grave need to find potential alternatives for sustainable poultry production. Piper betle (PB) and Persicaria odorata (PO) are herbs, which have been reported for antimicrobial, antioxidant, and anti-inflammatory properties. The present study aimed to estimate the influence of different dose supplementation of Piper betle leaf meal (PBLM) and Persicaria odorata leaf meal (POLM) on growth performance, ileal digestibility and gut morphology of broilers chickens. A total of 210 one day-old broiler chicks were randomly grouped into 7 treatments, and each treatment group has 3 replicates (n = 10) with a total number of 30 chicks. The treatments included T1 control (basal diet (BD) with no supplementation), T2 (BD + 2 g/kg PBLM); T3 (BD + 4 g/kg PBLM), T4 (BD + 8 g/kg PBLM), T5 (BD + 2 g/kg POLM), T6 (BD + 4 g/kg POLM), T7 (BD + 8 g/kg POLM). Growth performance, gut morphology and ileal digestibility were measured. Except for T4 (8 g/kg PBLM), graded dose inclusion of PBLM and POLM increased (P < 0.05) the body weight gain (BWG), positively modulated the gut architecture and enhanced nutrient digestibility in both stater and finisher growth phases of broiler chickens. Birds fed on PBLM 4 g/kg (T3), and POLM 8 g/kg (T7) had significantly higher (P < 0.05) BWG with superior (P < 0.05) feed efficiency in the overall growth period. Chickens fed on diets T3 and T7 had longer (P < 0.05) villi for duodenum as well as for jejunum. Furthermore, the birds fed on supplementations T3 and T7 showed improved (P < 0.05) digestibility of ether extract (EE), and dry matter (DM) compared to the control group. However, least (P < 0.05) crude protein (CP) digestibility was recorded for T4. In conclusion, dietary supplementations of PBLM 4 g/kg and POLM 8 g/kg were positively modulated the intestinal microarchitecture with enhanced nutrient digestibility, resulted in maximum body weight gain, thus improved the growth performance of broiler chickens.
Project description:T7-like bacteriophages are a class of virulent bacteriophages which have a clearer genetic background and smaller genomes than other phages. In addition, it grows faster and is easier to culture than other phages. At present, the numbers of available T7-like bacteriophage genomes and Stenotrophomonas maltophilia genomes are small, and IME15 is the first T7-like virulent Stenotrophomonas phage whose sequence has been reported. It shows effective lysis of S. maltophilia. Here we announce its complete genome, and major findings from its annotation are described.
Project description:Inaccuracies in biochemically characterizing the amount and CO2-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco (RsRubisco) mutant with improved CO2-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (kcatC, up 27%), carboxylation efficiency (kcatC/Km for CO2, increased 17%), unchanged CO2/O2 specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of RsRubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L8S8) assembly. An N-terminal Val-11-Ile substitution did not affect RsRubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on RsRubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility).
Project description:Use of bacteriophages as a therapy for bacterial infection has been attempted over the last century. Such an endeavor requires the elucidation of basic aspects of the host-virus interactions and the resistance mechanisms of the host. Two recently developed bacterial collections now enable a genomewide search of the genetic interactions between Escherichia coli and bacteriophages. We have screened >85% of the E. coli genes for their ability to inhibit growth of T7 phage and >90% of the host genes for their ability to be used by the virus. In addition to identifying all of the known interactions, several other interactions have been identified. E. coli CMP kinase is essential for T7 growth, whereas overexpression of the E. coli uridine/cytidine kinase inhibits T7 growth. Mutations in any one of nine genes that encode enzymes for the synthesis of the E. coli lipopolysaccharide receptor for T7 adsorption leads to T7 resistance. Selection of T7 phage that can recognize these altered receptors has enabled the construction of phage to which the host is 100-fold less resistant.
Project description:BACKGROUND: Kluyvera, a genus within the family Enterobacteriaceae, is an infrequent cause of human infections. Bacteriophage Kvp1, the only bacteriophage isolated for one of its species, Kluyvera cryocrescens, is a member of the viral family Podoviridae. RESULTS: The genome of Kvp1, the first Kluyvera cryocrescens-specific bacteriophage, was sequenced using pyrosequencing (454 technology) at the McGill University and Genome Québec Innovation Centre. The two contigs were closed using PCR and the sequence of the terminal repeats completed by primer walking off the phage DNA. The phage structural proteome was investigated by SDS-PAGE and mass spectrometry. CONCLUSION: At 39,472 bp, the annotated genome revealed a closer relationship to coliphage T3 than T7 with Kvp1 containing homologs to T3 early proteins S-adenosyl-L-methionine hydrolase (0.3) and protein kinase (0.7). The quantitative nature of the relationships between Kvp1 and the other members of the T7-like virus genus (T7, T3, phiA1122, phiYeO3-12, Berlin, K1F, VP4 and gh-1) was confirmed using CoreGenes.
Project description:A 3382 bp fragment containing a gene for a DNA ligase from the extremely thermophilic, acidophilic, and facultatively anaerobic archaeon (archaebacterium) Desulfurolobus ambivalens was cloned and sequenced. The deduced amino acid sequence (600 amino acids, 67619 molecular weight) showed 30-34% sequence identity with the ATP-dependent eucaryal (eukaryotic) DNA ligases of Schizosaccharomyces pombe, Saccharomyces cerevisiae, the human DNA ligase I, and with the Vaccinia DNA ligase. Distant similarity to the DNA ligases from the bacteriophages T3, T4, T6, T7 and the African swine fever virus was found, whereas no similarities were detectable to the NAD-dependent DNA ligases from the bacteria (eubacteria) Escherichia coli and Thermus thermophilus, to the ATP-dependent RNA-ligase of bacteriophage T4, and to the tRNA-Ligase from S.cerevisiae. A detailed comparison of the phylogenetic relationship of the amino acid sequences of all known DNA and RNA ligases is presented including a complete alignment of the ATP-dependent DNA ligases. The in vivo-transcription initiation and termination sites of the D.ambivalens gene were mapped. The calculated transcript length was 1904-1911 nt.