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PubMed2001

Of microbes, mice and man.

This chapter reviews my 18 years of research in Anne's Unit including studies on temporal and spatial aspects of X-chromosome inactivation and imprinting and the role of methylation in X-inactivation in these processes during female mouse embryo development. To enable molecular studies of embryos, we developed a plethora of single cell assays for specific enzyme activity, gene mutation and methylation, and RNA transcription. While in Anne's Unit, I used these same single cell assays to pioneer the procedures for preimplantation diagnosis of genetic disease, now an established clinical approach to prevention of the birth of children with severe genetic disease. At the Institute of Child Health in London, we continue to develop new highly sensitive molecular procedures--currently for the creation of cDNA libraries from human preimplantation embryos, primordial germ cells and embryonal stem cells. We are using these cDNA preparations to isolate human developmental genes and embryo/cancer genes. One of the more fascinating aspects arising from my time in Anne's Unit is the way in which my research findings challenged a number of accepted dogmas in development concerned with the origin and totipotency of the germ line and the possibility of transgenerational genetic inheritance by epigenetic modification of the germ line.

Animals↗
PubMed1999

Peptide nucleic acid (PNA) antisense effects in Escherichia coli.

Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake, antisense PNAs may find applications as antimicrobial agents and as tools for microbial functional genomics.

Antisense Elements (Genetics)↗
PubMed2002

Proteomics approaches towards antigen discovery and vaccine development.

Vaccines have proved to be powerful medical interventions, and recent advances in immunology and in microbial pathogen genomics now allow the rational design of molecularly defined vaccines. Classical proteomics and immunoproteomics approaches such as serological proteome analysis (SERPA) have already provided first vaccine candidate antigens. The same is true for approaches based on recombinant DNA technology, and in the future protein arrays may further accelerate this kind of research. Taken together, these technologies will allow identification of the full set of antigens (ie, the immunome) targeted by the immune system in a certain pathological situation. The comparison of multiple immunomes may allow the discovery of immunogenic structural features shared and conserved between different pathogens, which could form the basis of broadly protective vaccines. In this review, recent proteomics and other post-genomics approaches for the identification of antigens in infectious disease and cancer, as well as strategies for their characterization, are discussed.

Antigens↗
PubMedDate not supplied

[Typing of Salmonella by DNA-microarrays].

Microarrays (DNA-Chips) are miniaturized carriers on which many nucleic acid molecule probes such as oligonucleotides or PCR products are immobilized in a high density, and compactness. Homologue DNA hybridises with the immobilized complementary nucleic acid probes. This study gives after a short general introduction in the principle of DNA-microarrays an overview about published data on the field of typing of Salmonella by microarrays. An onset of a DNA-microarray developed by the National Reference Laboratory for Salmonella (NRL-Salm) will be introduced. By this new technique, it is possible to answer epidemiological questions as well as to find genes involved in certain biochemical processes, such as pathogenicity or resistance of salmonellae.

Animals↗
PubMed1993

Lipid hydroperoxide-resistance gene in Saccharomyces cerevisiae: utilization as a selectable marker gene for yeast transformation.

Two kinds of DNA fragments concerned with resistance against lipid hydroperoxide were cloned from the yeast Saccharomyces cerevisiae. When the recombinant plasmid carrying the DNA fragment was introduced into the cells of S. cerevisiae which do not have an appropriate auxotrophic marker, the transformants could be selected by their resistance to t-butyl hydroperoxide. By Southern hybridization analysis, such transformants were found to contain the recombinant plasmid, suggesting that the DNA fragment could be used as a selection marker for the transformation of S. cerevisiae.

Cloning, Molecular↗
PubMed2026

Catalog of metagenome-assembled genomes of prokaryotic communities from the Red Sea hydrothermal vents.

This study presents medium- and high-quality prokaryotic metagenome-assembled genomes (MAGs) from microbial mats and sediments at Hatiba Mons, a Red Sea hydrothermal system. We recovered 1,217 bacterial and archaeal MAGs across 75 phyla, dominated by Planctomycetota and Thermoproteota. Approximately 70% of these genomes likely represent previously uncharacterized taxa.

extreme environment↗
PubMed2026

Whole genome sequencing and phenotypic characterization of microbial isolates associated with two Floridian fig species.

Fig-associated microbes are increasingly recognized as important contributors to the ecology of fig ( Ficus ) communities, yet few cultured isolates are available for functional investigation. Students enrolled in a course-based undergraduate research experience (CURE) phenotypically characterized and generated high-quality genome assemblies for 22 bacterial isolates recovered from Ficus aurea and Ficus citrifolia in southern Florida, USA. These isolates represent six bacterial species spanning four genera and provide a genomic resource for future investigations of microbial interactions within fig communities.

Journal Article↗
PubMed2006

Microarray-based analysis of subnanogram quantities of microbial community DNAs by using whole-community genome amplification.

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (r2 = 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (r2 = 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r2 = 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment.

Biomass↗
PubMed2001

The Comprehensive Microbial Resource.

One challenge presented by large-scale genome sequencing efforts is effective display of uniform information to the scientific community. The Comprehensive Microbial Resource (CMR) contains robust annotation of all complete microbial genomes and allows for a wide variety of data retrievals. The bacterial information has been placed on the Web at http://www.tigr.org/CMR for retrieval using standard web browsing technology. Retrievals can be based on protein properties such as molecular weight or hydrophobicity, GC-content, functional role assignments and taxonomy. The CMR also has special web-based tools to allow data mining using pre-run homology searches, whole genome dot-plots, batch downloading and traversal across genomes using a variety of datatypes.

Bacteria↗
PubMed2026

Genome-resolved analysis reveals disruption of gut microbial vitamin B and K2 biosynthesis during Toxoplasma gondii infection in mice.

UNLABELLED: Toxoplasma gondii infection remodels the gut microbiome, yet its impact on microbial vitamin biosynthetic potential and host redox metabolism remains unclear. Here, we integrated mouse gut metagenomes with publicly available metagenome-assembled genomes (MAGs) to construct a genome-resolved atlas of B-vitamin and vitamin K2 biosynthesis. From 45,697 MAGs, we curated 4,771 representative genomes, of which 2,682 met high-quality criteria (completeness &#x2265;90%, contamination <5%). Functional annotation identified 229,717 vitamin-related genes corresponding to 177 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs across de novo pathways for eight B vitamins, thiamine (B1), riboflavin (B2), niacin (B3), pantothenate (B5), pyridoxine (B6), biotin (B7), folate (B9), cobalamin (B12), and vitamin K2. Among the high-quality genomes, 1,665 encoded complete de novo pathways for at least one vitamin, highlighting functional specialization and community-level complementarity. Transcripts per million-normalized metagenomic read counts revealed significant differences in KEGG ortholog abundances across six of the nine vitamin pathways. Reanalysis of metagenomic data from infected mice (acute, chronic, and control; n = 10 per group) revealed a stage-dependent reduction in &#x3b1;-diversity of vitamin biosynthesis pathways during acute infection, and a clear &#x3b2;-diversity separation from chronic and control groups. Core niacin biosynthesis genes (nadB, nadA, nadC) displayed phylum-specific redistribution, indicating selective remodeling of microbial NAD+ precursor production under infection-induced metabolic stress. These results suggest that T. gondii infection disrupts cooperative vitamin biosynthetic networks while specifically modulating niacin pathways linked to host NAD+ metabolism. IMPORTANCE: Gut microbes can synthesize essential vitamins, but how infection alters this function is poorly understood. By integrating mouse gut metagenomes with genome-resolved microbial data, we show that Toxoplasma gondii infection reshapes the vitamin biosynthetic potential of the gut microbiome in a stage-dependent manner. Acute infection reduces the diversity of vitamin biosynthesis pathways and shifts the taxonomic distribution of key niacin biosynthesis genes involved in microbial NAD+ precursor production. These findings identify vitamin metabolism, especially niacin-related pathways, as a sensitive functional axis of microbiome remodeling during infection. Our work links microbial taxonomic changes to functional metabolic consequences and suggests that microbiome-mediated regulation of NAD+-related metabolism may contribute to host redox adaptation during T. gondii infection.

B vitamins↗
PubMed2005

Evolution of a microbial nitrilase gene family: a comparative and environmental genomics study.

BACKGROUND: Completed genomes and environmental genomic sequences are bringing a significant contribution to understanding the evolution of gene families, microbial metabolism and community eco-physiology. Here, we used comparative genomics and phylogenetic analyses in conjunction with enzymatic data to probe the evolution and functions of a microbial nitrilase gene family. Nitrilases are relatively rare in bacterial genomes, their biological function being unclear. RESULTS: We examined the genetic neighborhood of the different subfamily genes and discovered conserved gene clusters or operons associated with specific nitrilase clades. The inferred evolutionary transitions that separate nitrilases which belong to different gene clusters correlated with changes in their enzymatic properties. We present evidence that Darwinian adaptation acted during one of those transitions and identified sites in the enzyme that may have been under positive selection. CONCLUSION: Changes in the observed biochemical properties of the nitrilases associated with the different gene clusters are consistent with a hypothesis that those enzymes have been recruited to a novel metabolic pathway following gene duplication and neofunctionalization. These results demonstrate the benefits of combining environmental genomic sampling and completed genomes data with evolutionary and biochemical analyses in the study of gene families. They also open new directions for studying the functions of nitrilases and the genes they are associated with.

Aminohydrolases↗
PubMed2005

Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis: genotyping single spores of arbuscular mycorrhizal (AM) fungi.

Limited biological samples of microbial origin often yield insufficient amounts of genomic DNA, making application of standard techniques of genetic analysis, like amplified fragment length polymorphism (AFLP), virtually impossible. The Phi29 DNA polymerase based whole genome amplification (WGA) method has the potential to alleviate this technical bottleneck. In the present work, we have sought to investigate the suitability of genomic DNA synthesized using Phi29 based WGA for AFLP analysis. We first used genomic DNA from Saccharomyces cerevisiae to optimize the protocol for the use of SDA-amplified DNA for AFLP analysis. Based on the optimized protocol we obtained AFLP fingerprints which were indistinguishable from the non-amplified genomic DNA. Finally, AFLP analysis was performed using SDA synthesized genomic DNA from single spores of various species of arbuscular mycorrhizal (AM) fungi. Unique and highly reproducible fingerprints for each species were obtained. The present study introduces the application of WGA-mediated AFLP to AM fungal biology; similarly, our protocol could be useful for other microbial genomes currently not amenable to genetic analysis owing to the paucity of starting template.

DNA Replication↗
PubMed2002

Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes.

Microsatellites are a ubiquitous class of simple repetitive DNA sequence. An excess of such repetitive tracts has been described in all eukaryotes analyzed and is thought to result from the mutational effects of replication slippage. Large-scale genomic and EST sequencing provides the opportunity to evaluate the abundance and relative distribution of microsatellites between transcribed and nontranscribed regions and the relationship of these features to haploid genome size. Although this has been studied in microbial and animal genomes, information in plants is limited. We assessed microsatellite frequency in plant species with a 50-fold range in genome size that is mostly attributable to the recent amplification of repetitive DNA. Among species, the overall frequency of microsatellites was inversely related to genome size and to the proportion of repetitive DNA but remained constant in the transcribed portion of the genome. This indicates that most microsatellites reside in regions pre-dating the recent genome expansion in many plants. The microsatellite frequency was higher in transcribed regions, especially in the untranslated portions, than in genomic DNA. Contrary to previous reports suggesting a preferential mechanism for the origin of microsatellites from repetitive DNA in both animals and plants, our findings show a significant association with the low-copy fraction of plant genomes.

Animals↗
PubMed2026

The chromosomal genome sequence of the spiny sea fan, Muricea muricata (Pallas, 1766) (Malacalcyonacea: Plexauridae) and its associated microbial metagenome sequences.

We present a genome assembly from a Muricea muricata specimen (spiny sea fan; Cnidaria; Anthozoa; Malacalcyonacea; Plexauridae). The genome sequence has a total length of 453.40 megabases. Most of the assembly (98.45%) is scaffolded into 16 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 19.29 kilobases. Gene annotation of this assembly by Ensembl identified 52 164 protein-coding genes. From the metagenome data, we recovered five bins, of which three were high-quality MAGs.

Malacalcyonacea↗
PubMed2006

Cloning of environmental genomic fragments as physical markers for monitoring microbial populations in coking wastewater treatment system.

The association between community functional shift and dynamics of genomic DNA composition can be used to identify functionally relevant populations as indicator organisms for systems monitoring. In this work, fingerprinting-based community DNA hybridization was used to monitor community structural dynamics and identify genomic fragments whose abundance shifts were concomitant to changes in COD removal capacity in a reactor. A laboratory-scale anaerobic-anoxic-oxic fixed biofilm system treating coking wastewater was operated with (LR mode) or without effluent recirculation (LNR mode). The contribution to total chemical oxygen demand (COD) removal by the anoxic reactor increased from 4% in LNR mode to 26% in LR mode. Long primer RAPD (randomly amplified polymorphic DNA) community fingerprints of the anoxic reactor also changed most significantly from the one similar to the anaerobic reactor to one similar to the oxic reactor. DNA hybridization revealed one signature band of 2.1 kb shared by the anoxic and oxic reactors in LR, but not LNR mode. Clone library profiling of this band resulted in one predominant 2.1-kb genomic fragment (B3) with no homologous sequences in GenBank. Real-time polymerase chain reaction indicated that copy numbers of B3 in the anoxic reactor under LR mode were 69 times higher than that under LNR mode, concomitant to a significant increase in COD removal capacity in this reactor. The different patterns of distribution of B3 in the laboratory system and a comparable malfunctioning industrial system demonstrated the potential of this genomic fragment as physical markers in systems monitoring. In addition, this genomic fragment may allow sequence-guided isolation of the host microbe.

Bacteria↗
PubMed2026

Lineage dynamics of invasive Escherichia coli isolates in the Netherlands from 1975 to 2021: a retrospective longitudinal genomic analysis.

BACKGROUND: Escherichia coli is a common cause of invasive infections such as bloodstream and cerebrospinal fluid infections in neonates. Strains positive for the K1 capsule are considered the most common cause of such neonatal invasive infections. This assumption of K1 dominance, and indeed the population genomics of E coli causing invasive infections in general is largely unstudied. We aimed to provide a comprehensive characterisation of this pathogen population using a longitudinal isolate collection. METHODS: In this analysis we report the findings of the SENTINEL study, a longitudinal genomic analysis of 1790 invasive E coli isolates collected mainly from newborns in the Netherlands between 1975 and 2021 by the Netherlands Reference Laboratory for Bacterial Meningitis, Amsterdam University Medical Centre, Amsterdam, Netherlands. The dataset included all bacterial strains cultured from cerebrospinal fluid or blood in cases of (clinical) bacterial meningitis (1976 to 1980). In 1981 the criteria were expanded to include neonates (aged &#x2264;4 weeks) with E coli sepsis, and from July, 2016 all infants younger than 1 year with E coli sepsis were included. All isolates were sequenced using either the HiSeq 2500 or HiSeq 4000 platforms (Illumina, San Diego, CA, USA). We confirmed species and identified sequence types (STs), detected antimicrobial resistance genes, virulence genes, and the presence of K1 capsule, and characterised the dynamics of these factors over time. FINDINGS: Our data show a highly dynamic bacterial population that is entirely unaffected by antimicrobial resistance determinants. Key pathogen population fluctuations include the complete disappearance of the dominant lineage ST567 and the swapping of dominant ST95 clones from a single serotype O18:H7 clone to two distinct serotype O1:H7 clones, with changes in virulence factors including major fimbrial adhesins. These findings, combined with only 58&#xb7;8% (1053 of 1790) prevalence in K1-expressing isolates in the entire study population, point to host-pathogen interaction and immune selection pressures as key drivers of bacterial population dynamics in this largely antimicrobial-naive population. INTERPRETATION: Our data show the vital need for ongoing genomic surveillance of microbial pathogen populations to guide appropriate intervention strategies. Additionally, genomic insights of a pathogen population from one specific disease syndrome or patient population cannot always be generalised across other cohorts. FUNDING: Wellcome Antimicrobial and Antimicrobial Resistance Doctoral Training Programme and the National Institute for Health and Care Research Birmingham Biomedical Research Centre.

Netherlands↗