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At least 631 records · Page 35Linked to original sources

PubMed2001

Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.

Attachment Sites, Microbiological↗

PubMed1995

Analysis of the insertion characteristics of Tn4351 during high frequency transposition of Porphyromonas gingivalis.

In this study, we characterized the high frequency transfer of the Bacteroides fragilis transposon Tn4351 into the chromosome of Porphyromonas gingivalis using a system developed by Genco, et al. Using this system, transconjugants were isolated and genomic sequences analyzed using both Ava-I and EcoR1 to determine the insertion characteristics of Tn4351 into the genome of P. gingivalis. Although previous evidence pointed to the possibility of random insertion, this study shows that, in fact, the insertion characteristics of Tn4351 into the genome of P. gingivalis are nonrandom and predictable.

Bacteroides fragilis↗

PubMed1999

David Hopwood and the emergence of Streptomyces genetics.

Streptomyces spp. are unusual among bacteria in growing as mycelial colonies with sporulating aerial hyphae. They are very important as the source of most of the major antibiotics. Pioneering work by David Hopwood in the 1950s and 1960s established Streptomyces coelicolor A3(2) as the model system for the genus. Since then he has led successive key phases of research on this organism. In the 1970s, plasmids were discovered and characterised, and used both to establish conditions for transformation and in the subsequent development of cloning vectors. Protoplasts were exploited in both transformation and highly efficient cell fusion. In the 1980s, the early cloning of resistance genes from antibiotic-producing strains was followed by the cloning of antibiotic biosynthetic gene clusters, and the development of general methods and probes for the cloning of such clusters from diverse species. Analysis of these gene sets led to wide-ranging inferences about the biosynthesis of the important polyketide class of antibiotics, and to the production of hybrid antibiotics, and then, in the last decade, to more sophisticated combinatorial biosynthesis of designer molecules. In parallel, David Hopwood's work has also provided a crucial platform for studies of the regulation of the morphological and physiological differentiation that is manifested by sporulating antibiotic-producing colonies. Most recently, his involvement in the physical mapping of the entire 8 Mb genome of S. coelicolor A3(2) has culminated in its complete DNA sequencing: a project that should be completed under his management during the year 2000.

Anti-Bacterial Agents↗

PubMed2003

An integrated genetic data environment (GDE)-based LINUX interface for analysis of HIV-1 and other microbial sequences.

MOTIVATION: Sequence databases encode a wealth of information needed to develop improved vaccination and treatment strategies for the control of HIV and other important pathogens. To facilitate effective utilization of these datasets, we developed a user-friendly GDE-based LINUX interface that reduces input/output file formatting. DESIGN AND RESULTS: GDE was adapted to the Linux operating system, bioinformatics tools were integrated with microbe-specific databases, and up-to-date GDE menus were developed for several clinically important viral, bacterial and parasitic genomes. Each microbial interface was designed for local access and contains Genbank, BLAST-formatted and phylogenetic databases. AVAILABILITY: GDE-Linux is available for research purposes by direct application to the corresponding author. Application-specific menus and support files can be downloaded from (http://www.bioafrica.net).

Database Management Systems↗

PubMed2026

Genome-wide association analysis reveals specialization to hosts and niches in multiple species of the Lactobacillaceae.

The Lactobacillaceae inhabit diverse environments, but the extent of their habitat adaptation remains unclear and the colonization factors unknown. First, we applied multiple machine learning models to determine if we can distinguish strains of the same species isolated from two different habitats based on their gene content. Surprisingly, we show that no species is differentially adapted to the oral cavity versus the human gut, or food versus the human gut, while only Lactobacillus crispatus showed specialization to the human urogenital system versus human gut. We then asked which species of Lactobacillaceae are habitat-specialized and how they could be identified. Using multiple lifestyle predictors incorporated in logistic regression models, we found that Limosilactobacillus reuteri, Ligilactobacillus ruminis, L. salivarius, L. crispatus, and L. mucosae displayed the highest degrees of host specialization. Applying our microbial genome-wide association study tool, aurora, to these species identified genes encoding adhesins and bacteriocins as the strongest and most common adaptation factors. This work establishes a generalizable framework for identifying novel species-habitat pairs with strong evidence of specialization and for uncovering the genomic features underlying within-species host and habitat adaptation.

Humans↗

PubMed2004

Functional genomics-based studies of the microbial ecology of hyperthermophilic micro-organisms.

Although much attention has been paid to the genetic, biochemical and physiological aspects of individual hyperthermophiles, how these unique micro-organisms relate to each other and to their natural habitats must be addressed in order to develop a comprehensive understanding of life at high temperatures. Phylogenetic 16 S rRNA-based profiling of samples from various geothermal sites has provided insights into community structure, but this must be complemented with efforts to relate metabolic strategies to biotic and abiotic characteristics in high-temperature habitats. Described here are functional genomics-based approaches, using cDNA microarrays, to gain insight into how ecological features such as biofilm formation, species interaction, and possibly even gene transfer may occur in native environments, as well as to determine what genes or sets of genes may be tied to environmental functionality.

Biofilms↗

PubMed2005

Community proteomics of a natural microbial biofilm.

Using genomic and mass spectrometry–based proteomic methods, we evaluated gene expression, identified key activities, and examined partitioning of metabolic functions in a natural acid mine drainage (AMD) microbial biofilm community. We detected 2033 proteins from the five most abundant species in the biofilm, including 48% of the predicted proteins from the dominant biofilm organism, Leptospirillum group II. Proteins involved in protein refolding and response to oxidative stress appeared to be highly expressed, which suggests that damage to biomolecules is a key challenge for survival. We validated and estimated the relative abundance and cellular localization of 357 unique and 215 conserved novel proteins and determined that one abundant novel protein is a cytochrome central to iron oxidation and AMD formation.

Amino Acids↗

PubMed2004

The Horizontal Gene Pool: an ESF workshop summary.

The European Science Foundation (ESF) funds a limited number of exploratory workshops each year that enable scientists to meet and develop plans for a program of integrated research which would benefit from a coordinated European effort. In summer 2003, the Standing Committee for Life and Environmental Sciences (LESC) sponsored such a workshop called The Horizontal Gene Pool: The Functional Role of Mobile Genetic Information--How Bacteria Perceive, Sample, and Utilize Genetic Elements in evolution and Local Adaptation. The workshop took place at St. Catherine's College, Oxford, UK. Its purpose was to identify how recent advances in the application of genomics and microbial ecology can be harnessed to determine the genetic mechanisms that underpin the biological role of the horizontal gene pool. Scientific excellence at the workshop was contributed by senior scientists and young investigators from research institutes located in nine European countries.

Bacteria↗

PubMed1998

Molecular biology of varicella-zoster virus. A review prepared for the UK Advisory Group on Chickenpox.

Varicella-zoster virus (human herpesvirus 3; VZV) is one of eight herpes viruses that routinely infect humans. It is classified as a member of the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae. Of the other human herpes viruses it is most closely related to the herpes simplex viruses (also members of the Alphalerpesvirinae). Like all herpes viruses, the virus has a large double-stranded DNA genome within an icosahedral nucleocapsid. This is surrounded by a proteinaceous tegument and a trilaminar membrane derived from host-cell membranes into which the viral glycoproteins are inserted. The structure of the virion is summarized in Fig. 1.

Antiviral Agents↗

PubMed2001

Bactericidal antisense effects of peptide-PNA conjugates.

Antisense peptide nucleic acids (PNAs) can specifically inhibit Escherichia coli gene expression and growth and hold promise as anti-infective agents and as tools for microbial functional genomics. Here we demonstrate that chemical modification improves the potency of standard PNAs. We show that 9- to 12-mer PNAs, especially when attached to the cell wall/membrane-active peptide KFFKFFKFFK, provide improvements in antisense potency in E. coli amounting to two orders of magnitude while retaining target specificity. Peptide-PNA conjugates targeted to ribosomal RNA (rRNA) and to messenger RNA (mRNA) encoding the essential fatty acid biosynthesis protein Acp prevented cell growth. The anti-acpP PNA at 2 microM concentration cured HeLa cell cultures noninvasively infected with E. coli K12 without any apparent toxicity to the human cells. These results indicate that peptides can be used to carry antisense PNA agents into bacteria. Such peptide-PNA conjugates open exciting possibilities for anti-infective drug development and provide new tools for microbial genetics.

Amino Acid Sequence↗

PubMed1993

Unrelatedness of multiply resistant Staphylococcus aureus with resistance to methicillin and to quinolones (QR-MRSA) as evident from SmaI-digestion patterns of genomic DNA.

Methicillin-resistant S. aureus with quinolone-resistance (QR-MRSA) isolated in Germany from three outbreaks of nosocomial infections and from sporadic nosocomial infections in five hospitals exhibited different SmaI-restriction patterns of their genomic DNA. Phage-typing and determination of plasmid profiles performed in parallel confirmed this differentiation, with one exception. These results indicate that there is obviously no overregional spread of one particular QR-MRSA clone and that quinolone resistance has developed independently in different MRSA.

Bacterial Typing Techniques↗

PubMed2026

Matching genomic evidence to claims about Mycobacterium avium subsp. paratuberculosis: Host association, host adaptation, mechanism, and virulence.

Whole-genome sequencing (WGS) permits high-resolution comparison of Mycobacterium avium subsp. paratuberculosis (MAP) isolates and pangenome analysis. Combined with animal-movement data, WGS can support transmission inference, but resolution alone does not establish the biological meaning of genomic variation. This focused narrative review applies a two-dimensional framework to purposively selected MAP studies, separating claim targets from support profiles. Claim targets include lineage identity, host-source or lineage characterization, host association, transmission, candidate genomic features, measured bacterial or host-cell phenotypes, natural-host infection fitness, disease or damage, shedding, and control outcomes. Depending on the claim, evidence operations may include characterization, context-aware comparative inference, direct endpoint ascertainment, and controlled feature perturbation; these are non-ordinal and may co-occur. On-target attribution, independent replication, and transportability are reported separately. The claim, not the study, is the unit of assessment. Typing markers support isolate or lineage discrimination, whereas phylogenomics supports evolutionary inference; neither alone establishes host adaptation. Pangenome comparisons and microbial genome-wide association studies nominate candidate features rather than establish adaptation. Cell-envelope and iron-associated studies support specified biochemical, transcriptional, or physiological phenotypes under defined conditions, while macrophage and calf models support only the endpoints measured. Annotated sequence variation alone nominates pathogenicity hypotheses. Across the illustrative studies selected here, MAP genomics most directly supported lineage classification, candidate discovery, measured bacterial phenotypes, bounded transmission inference, and natural-host infection-fitness claims. Claims about adaptation, mechanism, virulence, or control require endpoints and comparisons matched to the stated claim and model; feature-specific causal claims additionally require evidence linking the bacterial feature to the measured endpoint.

Claim boundaries↗

PubMed2006

Exploration and grading of possible genes from 183 bacterial strains by a common protocol to identification of new genes: Gene Trek in Prokaryote Space (GTPS).

A large number of complete microorganism genomes has been sequenced and submitted to the public database and then incorporated into our complete genome database, Genome Information Broker (GIB, http://gib.genes.nig.ac.jp/). However, when comparative genomics is carried out, researchers must be aware that there are protein-coding genes not confirmed by homology or motif search and that reliable protein-coding genes are missing. Therefore, we developed a protocol (Gene Trek in Prokaryote Space, GTPS) for finding possible protein-coding genes in bacterial genomes. GTPS assigns a degree of reliability to predicted protein-coding genes. We first systematically applied the protocol to the complete genomes of all 123 bacterial species and strains that were publicly available as of July 2003, and then to those of 183 species and strains available as of September 2004. We found a number of incorrect genes and several new ones in the genome data in question. We also found a way to estimate the total number of orthologous genes in the bacterial world.

Bacteria↗

PubMed2006

454 sequencing put to the test using the complex genome of barley.

BACKGROUND: During the past decade, Sanger sequencing has been used to completely sequence hundreds of microbial and a few higher eukaryote genomes. In recent years, a number of alternative technologies became available, among them adaptations of the pyrosequencing procedure (i.e. "454 sequencing"), promising an approximately 100-fold increase in throughput over Sanger technology--an advancement which is needed to make large and complex genomes more amenable to full genome sequencing at affordable costs. Although several studies have demonstrated its potential usefulness for sequencing small and compact microbial genomes, it was unclear how the new technology would perform in large and highly repetitive genomes such as those of wheat or barley. RESULTS: To study its performance in complex genomes, we used 454 technology to sequence four barley Bacterial Artificial Chromosome (BAC) clones and compared the results to those from ABI-Sanger sequencing. All gene containing regions were covered efficiently and at high quality with 454 sequencing whereas repetitive sequences were more problematic with 454 sequencing than with ABI-Sanger sequencing. 454 sequencing provided a much more even coverage of the BAC clones than ABI-Sanger sequencing, resulting in almost complete assembly of all genic sequences even at only 9 to 10-fold coverage. To obtain highly advanced working draft sequences for the BACs, we developed a strategy to assemble large parts of the BAC sequences by combining comparative genomics, detailed repeat analysis and use of low-quality reads from 454 sequencing. Additionally, we describe an approach of including small numbers of ABI-Sanger sequences to produce hybrid assemblies to partly compensate the short read length of 454 sequences. CONCLUSION: Our data indicate that 454 pyrosequencing allows rapid and cost-effective sequencing of the gene-containing portions of large and complex genomes and that its combination with ABI-Sanger sequencing and targeted sequence analysis can result in large regions of high-quality finished genomic sequences.

Base Pairing↗

PubMed2004

[Strategies for bacterial virulence genes identification].

One of the recent preoccupations of medical microbiology has been to characterise the mechanisms of virulence of bacterial pathogens at the molecular level. One hundred years after Koch, Stanley Falkow proposed a new, molecular version of Koch's postulates to define avirulence gene: (a) the gene confers a certain phenotype to the studied bacteria, (b) inactivation of the gene abolishes the phenotype, (c)reintroduction of the gene restores the wild type to the mutant. Although this strategy, based upon mutagenesis and the use of experimental models, allows the identification of many genes, it is not comprehensive. Other methods can be used to complete the identification of virulence factors such as differential expression, either at the level of transcription (transcriptome) or at the level of protein expression (proteome). All these techniques are now supported by the data from complete genome sequencing projects. The pool of information obtained from these approaches allows the definition of the 'virulome', which is the assembly of factors a pathogen requires for virulence. Understanding the virulome will open the way to the development of new strategies for vaccination or the development of new generation of antimicrobials.

Animals↗

PubMed1994

On small genomes in eukaryotic organisms: molecular karyotypes of two microsporidian species (Protozoa) parasites of vertebrates.

Pulsed field gel electrophoresis (PFGE) was used to separate chromosome-sized DNA from two species of microsporidia of fishes. The molecular karyotype of Glugea atherinae exhibits 16 DNA bands from 420 to 2,700 kb, and that of Spraguea lophii 12 bands from 230 to 980 kb. Until now they represent respectively the largest and the smallest genomes visualized for microsporidia: 19.5 Mb for G. atherinae and 6.2 Mb for S. lophii (the smallest nuclear genome in eukaryotic organism). We have analysed separately five strains of G. atherinae (individual cysts), with this technique. The electrophoretic spectra are the same for these strains, except for the absence of the 2,380-kb band in one case. Therefore, the karyotype seems to be rather well conserved for this species.

Animals↗

PubMed1998

Tuberculosis one century after R. Koch.

The molecular revolution is beginning to have an impact on all aspects of tuberculosis. Work in diagnostics is expected to progress in maximizing genetic testing of mycobacteria. Drug development will benefit from recently acquired knowledge on molecular mechanisms of drug action. The availability of the complete genome of Mycobacterium tuberculosis, and better tools for genetic work with mycobacteria, will promote the development of vaccine candidates. Our work has contributed to the definition of the mechanisms of resistance to rifampin, fluoroquinolones, and ethambutol, and, using molecular analysis, to a better understanding of the epidemiology of mycobacterial infections. This overview of the direction of tuberculosis research today illustrates the impact of novel scientific approaches on the investigation of old problems.

Antitubercular Agents↗

PubMed2003

Evolution of glutamate dehydrogenase genes: evidence for lateral gene transfer within and between prokaryotes and eukaryotes.

BACKGROUND: Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH), to evaluate and compare the patterns and rates of lateral gene transfer (LGT) in prokaryotes and eukaryotes. RESULTS: We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists). CONCLUSION: LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

Algal Proteins↗