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PubMed2000

Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model.

A systematic method was developed to assemble functional full-length genomes of large RNA and DNA viruses. Coronaviruses contain the largest single-stranded positive-polarity RNA genome in nature. The approximately 30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an important pathogen in swine. Using a novel approach, six adjoining cDNA subclones that span the entire TGEV genome were isolated. Each clone was engineered with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of approximately 28.5 kb in length. Transcripts derived from the full-length TGEV construct were infectious, and progeny virions were serially passaged in permissive host cells. Viral antigen production and subgenomic mRNA synthesis were evident during infection and throughout passage. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar plaque morphology in permissive host cells. Host range phenotypes of the molecularly cloned and wild-type viruses were similar in cells of swine and feline origin. The recombinant viruses were sequenced across the unique interconnecting junctions, conclusively demonstrating the marker mutations and restriction sites that were engineered into the component clones. Full-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of TGEV could theoretically be used to precisely reconstruct microbial or eukaryotic genomes approaching several million base pairs in length.

Animals↗

PubMed2026

MetagenomicKG: a knowledge graph for metagenomic applications.

MOTIVATION: The sheer volume and variety of genomic content within microbial communities makes metagenomics a field rich in biomedical knowledge. To traverse these complex communities and their vast unknowns, metagenomic studies often depend on distinct reference databases, such as the Genome Taxonomy Database (GTDB), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), for various analytical purposes. These databases are crucial for the genetic and functional annotation of microbial communities. Nevertheless, the inconsistent nomenclature or identifiers of these databases present challenges for effective integration, representation, and utilization. Knowledge graphs (KGs) offer an appropriate solution by organizing biological entities from different databases to standardized identifiers, allowing their interrelations to be captured into a cohesive network regardless of the naming conventions used in each source. The graph structure not only facilitates the unveiling of hidden patterns but also enriches our biological understanding with deeper insights. Despite KGs having shown potential in various biomedical fields, their application in metagenomics remains underexplored. RESULTS: We present MetagenomicKG, a novel knowledge graph specifically tailored for metagenomic analysis. MetagenomicKG integrates taxonomic, functional, and pathogenesis-related information on the human microbiome sourced from various databases, and further connects these with existing biomedical KGs to expand the biological network. Through various case studies involving the human microbiome, we demonstrate its utility in enabling hypothesis generation regarding the relationships between microbes and diseases, generating sample-specific graph embeddings, and providing robust pathogen prediction. CODE AVAILABILITY: The source code and technical details for constructing the MetagenomicKG and reproducing all analyses are available on GitHub at https://github.com/KoslickiLab/MetagenomicKG. The data used in this manuscript, including the pre-built files and use case input data, are archived on Zenodo with DOI: 10.5281/zenodo.17546861.

Metagenomics↗

PubMed2026

A Strain of Mycoplasma hominis Causing Pleuropneumonia Infection in an Immunocompetent Patient and Recent Epidemiological Trends: Implications for Clinical Management.

Mycoplasma hominis (M. hominis) is an opportunistic pathogen linked to urogenital and neonatal infections; however, limited genetic and epidemiological data are available. Extragenital infections in healthy adults are rare, and effective antibiotics are species-specific, complicating diagnosis and treatment. Hence, this study aimed to elucidate the clinical process, update the epidemiological characteristics, and investigate the genomic features of a fluoroquinolone-resistant M. hominis isolate from an immunocompetent patient with pleuropneumonia in China. The M. hominis isolate ZY_MH01 was recovered from pleural fluid and was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Whole Genome Sequencing (WGS). The completed genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Snippy v4.4.5 was utilized to conduct a core genome single nucleotide polymorphism (cgSNP) analysis between ZY_MH01 and 144 M. hominis strains from the NCBI GenBank database. Subsequently, phylogenies were constructed using IQ-TREE v3.1.2 and visualized by iTOL. Antimicrobial resistance determinants were identified using strict criteria of Comprehensive Antibiotic Resistance Database (CARD) RGI 6.0.5 (Web portal) and broth microdilution test. Virulence genes were screened using Abricate v1.0.1 against the Virulence Factor Database (VFDB). A total of 42 strains (including ZY_MH01) from Genbank with an assembly level of Complete or Chromosome were re-annotated using Prokka v1.2.0 and pangenome analysis was performed using the Roary. The core genome constitutes only 17.1%, which may contribute to the unusually high level of polymorphism observed among M. hominis strains. No antibiotic resistance genes or virulence genes were detected in the genome of ZY_MH01. However, some resistance-associated mutations of gene parC and gyrA in the Quinolone Resistance Determining Region (QRDR) were identified. Phylogenetic analysis indicates that strains originating from the same geographic region typically exhibit reduced genetic distances; this trend is especially pronounced in regions with a higher number of publicly available strain sequences. In specific circumstances, when conventional broad-spectrum antibiotics are ineffective, even immunocompetent patients should consider the possibility of M. hominis infection. This study presents a detailed account of the diagnostic and therapeutic course of a pleuropneumonia infection caused by M. hominis in an immunocompetent patient, and performs an epidemiological analysis of all M. hominis sequences that have been recently made publicly available.

Humans↗

PubMed2004

Trends between gene content and genome size in prokaryotic species with larger genomes.

Although the evolution process and ecological benefits of symbiotic species with small genomes are well understood, these issues remain poorly elucidated for free-living species with large genomes. We have compared 115 completed prokaryotic genomes by using the Clusters of Orthologous Groups database to determine whether there are changes with genome size in the proportion of the genome attributable to particular cellular processes, because this may reflect both cellular and ecological strategies associated with genome expansion. We found that large genomes are disproportionately enriched in regulation and secondary metabolism genes and depleted in protein translation, DNA replication, cell division, and nucleotide metabolism genes compared to medium- and small-sized genomes. Furthermore, large genomes do not accumulate noncoding DNA or hypothetical ORFs, because the portion of the genome devoted to these functions remained constant with genome size. Traits other than genome size or strain-specific processes are reflected by the dispersion around the mean for cell functions that showed no correlation with genome size. For example, Archaea had significantly more genes in energy production, coenzyme metabolism, and the poorly characterized category, and fewer in cell membrane biogenesis and carbohydrate metabolism than Bacteria. The trends we noted with genome size by using Clusters of Orthologous Groups were confirmed by our independent analysis with The Institute for Genomic Research's Comprehensive Microbial Resource and Kyoto Encyclopedia of Genes and Genomes' Orthology annotation databases. These trends suggest that larger genome-sized species may dominate in environments where resources are scarce but diverse and where there is little penalty for slow growth, such as soil.

Animals↗

PubMed2006

CGHScan: finding variable regions using high-density microarray comparative genomic hybridization data.

BACKGROUND: Comparative genomic hybridization can rapidly identify chromosomal regions that vary between organisms and tissues. This technique has been applied to detecting differences between normal and cancerous tissues in eukaryotes as well as genomic variability in microbial strains and species. The density of oligonucleotide probes available on current microarray platforms is particularly well-suited for comparisons of organisms with smaller genomes like bacteria and yeast where an entire genome can be assayed on a single microarray with high resolution. Available methods for analyzing these experiments typically confine analyses to data from pre-defined annotated genome features, such as entire genes. Many of these methods are ill suited for datasets with the number of measurements typical of high-density microarrays. RESULTS: We present an algorithm for analyzing microarray hybridization data to aid identification of regions that vary between an unsequenced genome and a sequenced reference genome. The program, CGHScan, uses an iterative random walk approach integrating multi-layered significance testing to detect these regions from comparative genomic hybridization data. The algorithm tolerates a high level of noise in measurements of individual probe intensities and is relatively insensitive to the choice of method for normalizing probe intensity values and identifying probes that differ between samples. When applied to comparative genomic hybridization data from a published experiment, CGHScan identified eight of nine known deletions in a Brucella ovis strain as compared to Brucella melitensis. The same result was obtained using two different normalization methods and two different scores to classify data for individual probes as representing conserved or variable genomic regions. The undetected region is a small (58 base pair) deletion that is below the resolution of CGHScan given the array design employed in the study. CONCLUSION: CGHScan is an effective tool for analyzing comparative genomic hybridization data from high-density microarrays. The algorithm is capable of accurately identifying known variable regions and is tolerant of high noise and varying methods of data preprocessing. Statistical analysis is used to define each variable region providing a robust and reliable method for rapid identification of genomic differences independent of annotated gene boundaries.

Algorithms↗

PubMed2006

Protein repertoire of double-stranded DNA bacteriophages.

The complexity and diversity of phage gene sets, which are produced by rapid evolution of phage genomes and rampant gene exchanges among phages, hamper the efforts to decipher the evolutionary relationships between individual phage proteins and reconstruct the complete set of evolutionary events leading to the known phages. To start unraveling the natural history of phages, we built the phage orthologous groups (POGs), a natural system of phage protein families that includes 6378 genes from 164 complete genome sequences of double-stranded DNA bacteriophages. Phage proteomes have high POG coverage: on average, 39 genes per phage genome belong to POGs, which is close to half of all genes in most phages. In an agreement with the notion of phage role in horizontal gene transfer, we see many cases of likely gene exchange between phages and their microbial hosts. At the same time, about 80% of all POGs are highly specific to phage genomes and are not commonly found in microbial genomes, indicating coherence and large degree of evolutionary independence of phage gene sets. The information on orthologous genes is essential for evolutionary classification of known bacteriophages and for reconstruction of ancestral phage genomes.

Bacteriophages↗

PubMed2004

[Study on space flight-mutants of Monascus purpureus using Random Amplified Polymorphic DNA].

OBJECTIVE: To study the mechanism of microbial mutation in aerospace. METHOD: Random Amplified Polymorphic DNA (RAPD) was used to analyze the polymorphism of high Lovastatin-producing mutants. RESULT: Variations occurred in genomes of these mutants. CONCLUSION: The high Lovastatin producibility of the mutants is due to the variation of genomes, therefore the microbial mutation in the aerospace is achieved via the variation of hereditary genes in the microbes.

DNA, Fungal↗

PubMed2004

The genome revolution in vaccine research.

The conventional approach to vaccine development is based on dissection of the pathogen using biochemical, immunological and microbiological methods. Although successful in several cases, this approach has failed to provide a solution to prevent several major bacterial infections. The availability of complete genome sequences in combination with novel advanced technologies, such as bioinformatics, microarrays and proteomics, have revolutionized the approach to vaccine development and provided a new impulse to microbial research. The genomic revolution allows the design of vaccines starting from the prediction of all antigens in silico, independently of their abundance and without the need to grow the pathogen in vitro. This new genome-based approach, which we have named "Reverse Vaccinology", has been successfully applied for Neisseria meningitidis serogroup B for which conventional strategies have failed to provide an efficacious vaccine. The concept of "Reverse Vaccinology" can be easily applied to all the pathogens for which vaccines are not yet available and can be extended to parasites and viruses.

Bacterial Proteins↗

PubMed2000

Expression of secretory group IIA phospholipase A(2) in relation to the presence of microbial agents, macrophage infiltrates, and transcripts of proinflammatory cytokines in human aortic tissues.

Recent seroepidemiological and immunohistochemical studies have demonstrated an association between microbial infections and atherosclerosis. However, the mechanisms underlying this association are widely unknown. In the present study, arterial specimens obtained at autopsy after sudden death were analyzed concerning (1) the presence of Chlamydia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori; (2) the expression of secretory group IIA phospholipase A(2) (sPLA(2)-IIA) and of proinflammatory cytokines; and (3) the stage of atherosclerosis. Genomic DNA of microbial pathogens was determined by the polymerase chain reaction technique. The expression of sPLA(2)-IIA was studied immunohistochemically by using monoclonal antibodies against human sPLA(2)-IIA. Transcripts specific for sPLA(2)-IIA, interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were identified by reverse transcription-polymerase chain reaction. In 18 of 102 analyzed specimens, DNA of microbial pathogens was found. Thirteen sections were positive for C pneumoniae, whereas 2 specimens were positive either for cytomegalovirus or for herpes simplex virus. One section contained genomic DNA of all 3 pathogens simultaneously. None of the analyzed tissues exhibited nucleic acids specific for H pylori. In addition to macrophage infiltrates, the presence of microbial DNA was closely associated with the occurrence of transcripts specific for proinflammatory cytokines and sPLA(2)-IIA. Pathogens as well as sPLA(2)-IIA and cytokines were found to be present not only in advanced but also in early stages of atherosclerosis. In tissues negative for sPLA(2)-IIA and cytokine expression, none of the pathogens could be identified. Because macrophages exposed to phospholipase A(2)-treated lipoproteins are transformed into foam cells in vitro, the results of this study suggest an alternative mechanism by which microbial infections may act in a proatherogenic fashion in vessel walls.

Adolescent↗

PubMed2003

The uncultured microbial majority.

Since the delineation of 12 bacterial phyla by comparative phylogenetic analyses of 16S ribosomal RNA in 1987 knowledge of microbial diversity has expanded dramatically owing to the sequencing of ribosomal RNA genes cloned from environmental DNA. Currently, only 26 of the approximately 52 identifiable major lineages, or phyla, within the domain Bacteria have cultivated representatives. Evidence from field studies indicates that many of the uncultivated phyla are found in diverse habitats, and some are extraordinarily abundant. In some important environments, including seawater, freshwater, and soil, many biologically and geochemically important organisms are at best only remotely related to any strain that has been characterized by phenotype or by genome sequencing. Genome sequence information that would allow ribosomal RNA gene trees to be related to broader patterns in microbial genome evolution is scant, and therefore microbial diversity remains largely unexplored territory.

DNA, Bacterial↗

PubMed2001

Role of genomic typing in taxonomy, evolutionary genetics, and microbial epidemiology.

Currently, genetic typing of microorganisms is widely used in several major fields of microbiological research. Taxonomy, research aimed at elucidation of evolutionary dynamics or phylogenetic relationships, population genetics of microorganisms, and microbial epidemiology all rely on genetic typing data for discrimination between genotypes. Apart from being an essential component of these fundamental sciences, microbial typing clearly affects several areas of applied microbiological research. The epidemiological investigation of outbreaks of infectious diseases and the measurement of genetic diversity in relation to relevant biological properties such as pathogenicity, drug resistance, and biodegradation capacities are obvious examples. The diversity among nucleic acid molecules provides the basic information for all fields described above. However, researchers in various disciplines tend to use different vocabularies, a wide variety of different experimental methods to monitor genetic variation, and sometimes widely differing modes of data processing and interpretation. The aim of the present review is to summarize the technological and fundamental concepts used in microbial taxonomy, evolutionary genetics, and epidemiology. Information on the nomenclature used in the different fields of research is provided, descriptions of the diverse genetic typing procedures are presented, and examples of both conceptual and technological research developments for Escherichia coli are included. Recommendations for unification of the different fields through standardization of laboratory techniques are made.

Bacteria↗

PubMed2000

Will genome detection replace serology in blood screening for microbial agents?

The residual risk of transfusion-transmitted viral infection in developed countries is considered minimal or negligible. However, zero risk remains a strong political objective. Genomic screening for HCV, HIV and HBV represents a major advance, eliminating infectious blood donations collected during the pre-seroconversion window period, rare cases of immunosilent infections and, possibly, a large spectrum of viral variants. In Western countries, HCV RNA genomic screening started on pools of 16-400 plasma samples from individual donations. Pooling may produce false-positive and false-negative results. Individual donation testing is more suitable to blood screening but requires multiplexing, automation, and affordable cost. Because donations from individuals who are HBV DNA-negative/serologically positive, or those apparently recovered from HCV infection, may remain infectious, it is unlikely that HBsAg, anti-HCV, and anti-HIV will be discontinued when genomic screening is extended to all three viruses. HIV-1 p24 antigen may prove redundant with HIV RNA screening. Anti-HTLV-I and HTLV-II will remain more effective than genomic testing.

Antibodies, Viral↗

PubMed1999

Microbial gene transfer: an ecological perspective.

Microbial gene transfer or microbial sex is a means of exchanging loci amongst prokaryotes and certain eukaryotes. Historically viewed as a laboratory artifact, recent evidence from natural populations as well as genome research has indicated that this process may be a major driving force in microbial evolution. Studies with natural populations have taken two approaches- either adding a defined donor with a traceable gene to an indigenous community, and detecting the target gene in the indigenous bacteria, or by adding a model recipient to capture genes being transferred from the ambient microbial flora. However, both approaches usually require some cultivation of the recipient, which may result in a dramatic underestimation of the ambient transfer frequency. Novel methods are just evolving to study in situ gene transfer processes, including the use of green fluorescent protein (GFP)-marked plasmids, which enable detection of transferrants by epifluorescence microscopy. A transduction-like mechanism of transfer from viral-like particles produced by marine bacteria and thermal spring bacteria to Escherichia coil has been documented recently, indicating that broad host range transduction may be occurring in aquatic environments. The sequencing of complete microbial genomes has shown that they are a mosaic of ancestral chromosomal genes interspersed with recently transferred operons that encode peripheral functions. Archaeal genomes indicate that the genes for replication, transcription, and translation are all eukaryotic in complexity, while the genes for intermediary metabolism are purely bacterial. And in eukaryotes, many ancestral eukaryotic genes have been replaced by bacterial genes believed derived from food sources. Collectively these results indicate that microbial sex can result in the dispersal of loci in contemporary microbial populations as well as having shaped the phylogenies of microbes from multiple, very early gene transfer events.

Conjugation, Genetic↗

PubMed1997

Effect of nitrate injection on the microbial community in an oil field as monitored by reverse sample genome probing.

The reverse sample genome probe (RSGP) method, developed for monitoring the microbial community in oil fields with a moderate subsurface temperature, has been improved by (i) isolation of a variety of heterotrophic bacteria and inclusion of their genomes on the oil field master filter and (ii) use of phosphorimaging technology for the rapid quantitation of hybridization signals. The new master filter contains the genomes of 30 sulfate-reducing, 1 sulfide-oxidizing, and 16 heterotrophic bacteria. Most have been identified by partial 16S rRNA sequencing. Use of improved RSGP in monitoring the effect of nitrate injection in an oil field indicated that the sulfide-oxidizing, nitrate-reducing isolate CVO (a Campylobacter sp.) becomes the dominant community component immediately after injection. No significant enhancement of other community members, including the sulfate-reducing bacteria, was observed. The elevated level of CVO decayed at most sampling sites within 30 days after nitrate injection was terminated. Chemical analyses indicated a corresponding decrease and subsequent increase in sulfide concentrations. Thus, transient injection of a higher potential electron acceptor into an anaerobic subsurface system can have desirable effects (i.e., reduction of sulfide levels) without a permanent adverse influence on the resident microbial community.

Journal Article↗

PubMed2004

Mapping of microbial pathways through constrained mapping of orthologous genes.

We present a novel computer algorithm for mapping biological pathways from one prokaryotic genome to another. The algorithm maps genes in a known pathway to their homologous genes (if any) in a target genome that is most consistent with (a) predicted orthologous gene relationship, (b) predicted operon structures, and (c) predicted co-regulation relationship of operons. Mathematically, we have formulated this problem as a constrained minimum spanning tree problem (called a Steiner network problem), and demonstrated that this formulation has the desired property through applications. We have solved this mapping problem using a combinatorial optimization algorithm, with guaranteed global optimality. We have implemented this algorithm as a computer program, called PMAP. Our test results on pathway mapping are highly encouraging -- we have mapped a number of pathways of H. influenzae, B. subtilis, H. pylori, and M. tuberculosis to E. coli using P-MAP, whose homologous pathways in E coli. are known and hence the mapping accuracy could be checked. We have then mapped known E. coli pathways in the EcoCyc database to the newly sequenced organism Synechococcus sp WH8102, and predicted 158 Synechococcus pathways. Detailed analyses on the predicted pathways indicate that P-MAP's mapping results are consistent with our general knowledge about (local) pathways. We believe that P-MAP will be a useful tool for microbial genome annotation projects and inference of individual microbial pathways.

Algorithms↗

PubMed2005

A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus.

BACKGROUND: DNA microarray technology is widely used to determine the expression levels of thousands of genes in a single experiment, for a broad range of organisms. Optimal design of immobilized nucleic acids has a direct impact on the reliability of microarray results. However, despite small genome size and complexity, prokaryotic organisms are not frequently studied to validate selected bioinformatics approaches. Relying on parameters shown to affect the hybridization of nucleic acids, we designed freely available software and validated experimentally its performance on the bacterial pathogen Staphylococcus aureus. RESULTS: We describe an efficient procedure for selecting 40-60 mer oligonucleotide probes combining optimal thermodynamic properties with high target specificity, suitable for genomic studies of microbial species. The algorithm for filtering probes from extensive oligonucleotides libraries fitting standard thermodynamic criteria includes positional information of predicted target-probe binding regions. This algorithm efficiently selected probes recognizing homologous gene targets across three different sequenced genomes of Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset of control Escherichia coli probes was validated for applications in the fields of comparative genomics and molecular epidemiology, mapping of deletion mutations and transcription profiling. CONCLUSION: This generic chip-design process merging sequence information from several related genomes improves genome coverage even in conserved regions.

Algorithms↗

PubMed2005

DING proteins are from Pseudomonas.

DING proteins have been described as animal and plant proteins with potential biomineralisation, receptor or signalling roles that have been characterised by an N-terminal DINGGG-sequence. However, these sequences have only ever been identified as either N-terminal peptides or partial cDNA sequences, and have yet to be detected in any of the many genomic animal and plant genomes now available. Microbial relatives of the DING proteins have been described, which appear to be periplasmic phosphate-binding proteins. Recently, full-length Pseudomonas aeruginosa UCBPP-PA14 and Hypericum perforatum genes have been sequenced that show high homology to the published DING protein N-terminal sequences, and small peptides previously identified in conjunction with the peptide sequencing of DING proteins can also be mapped to regions across these full-length sequences. Searching with these sequences identifies other plant and animal cDNA fragments in the public nucleotide databases, and, additionally, an unordered rat genomic contig that contains a DING-like sequence on a small fragment. Analysing the codon usage of these DNA sequences identifies all of these sequences as of Pseudomonas origin, suggesting that DING proteins do not exist in eukaryotes, but instead are potentially due to microbial contamination or infection.

Amino Acid Sequence↗

PubMed2002

Principles of microbial alchemy: insights from the Streptomyces coelicolor genome sequence.

The world's most creative producers of natural pharmaceutical compounds are soil-dwelling bacteria classified as Streptomyces. The availability of the recently completed Streptomyces coelicolor genome sequence provides a link between the folklore of antibiotics and other bioactive compounds to underlying biochemical, molecular genetic and evolutionary principles.

Anti-Bacterial Agents↗