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PubMed2001

The University of Minnesota Biocatalysis/Biodegradation Database: emphasizing enzymes.

The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.ahc.umn.edu/) provides curated information on microbial catabolic enzymes and their organization into metabolic pathways. Currently, it contains information on over 400 enzymes. In the last year the enzyme page was enhanced to contain more internal and external links; it also displays the different metabolic pathways in which each enzyme participates. In collaboration with the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology, 35 UM-BBD enzymes were assigned complete EC codes during 2000. Bacterial oxygenases are heavily represented in the UM-BBD; they are known to have broad substrate specificity. A compilation of known reactions of naphthalene and toluene dioxygenases were recently added to the UM-BBD; 73 and 108 were listed respectively. In 2000 the UM-BBD is mirrored by two prestigious groups: the European Bioinformatics Institute and KEGG (the Kyoto Encyclopedia of Genes and Genomes). Collaborations with other groups are being developed. The increased emphasis on UM-BBD enzymes is important for predicting novel metabolic pathways that might exist in nature or could be engineered. It also is important for current efforts in microbial genome annotation.

Bacteria↗

PubMed2006

Bacterial populations as perfect gases: genomic integrity and diversification tensions in Helicobacter pylori.

Microorganisms that persist in single hosts face particular challenges. Helicobacter pylori, an obligate bacterial parasite of the human stomach, has evolved a lifestyle that features interstrain competition and intraspecies cooperation, both of which involve horizontal gene transfer. Microbial species must maintain genomic integrity, yet H. pylori has evolved a complex nonlinear system for diversification that exists in dynamic tension with the mechanisms for ensuring fidelity. Here, we review these tensions and propose that they create a dynamic pool of genetic variants that is sufficiently genetically diverse to allow H. pylori to occupy all of the potential niches in the stomach.

Biological Evolution↗

PubMed2005

Toward a genome-wide systems biology analysis of host-pathogen interactions in group A Streptococcus.

Genome-wide analysis of microbial pathogens and molecular pathogenesis processes has become an area of considerable activity in the last 5 years. These studies have been made possible by several advances, including completion of the human genome sequence, publication of genome sequences for many human pathogens, development of microarray technology and high-throughput proteomics, and maturation of bioinformatics. Despite these advances, relatively little effort has been expended in the bacterial pathogenesis arena to develop and use integrated research platforms in a systems biology approach to enhance our understanding of disease processes. This review discusses progress made in exploiting an integrated genome-wide research platform to gain new knowledge about how the human bacterial pathogen group A Streptococcus causes disease. Results of these studies have provided many new avenues for basic pathogenesis research and translational research focused on development of an efficacious human vaccine and novel therapeutics. One goal in summarizing this line of study is to bring exciting new findings to the attention of the investigative pathology community. In addition, we hope the review will stimulate investigators to consider using analogous approaches for analysis of the molecular pathogenesis of other microbes.

Computational Biology↗

PubMed2026

Nonantibiotic-driven evolution reveals rare but predictable routes to broad antibiotic resistance.

Many medications not prescribed to treat infectious diseases have antibacterial activity at physiologically relevant concentrations, raising the risk that chronic administration of such nonantibiotics may inadvertently select for resistance in the host microbiome. However, how frequently such exposures select for adaptations that impact broad drug resistance, including to antibiotics, remains unclear. Here, we systematically evolved Escherichia coli under exposure to 40 antibiotics and nonantibiotics and profiled the cross-resistance of the drug-adapted strains to 21 antibiotics representing all major classes. Our measurements revealed that most drug-adapted strains did not become multidrug resistant. However, five nonantibiotics and three antibiotics emerged as exceptions and were repeatedly selected for broad antibiotic resistance. Whole-genome sequencing of all 168 evolved strains revealed that changes in the regulation of efflux pumps repeatedly underlay broad drug resistance and converged into two key regulatory genes, acrR and lon. Our work suggests that although inadvertent antibiotic cross-resistance is rare, specific nonantibiotics can still potentially pose a risk for the emergence of multidrug resistance.IMPORTANCEMany medications not typically prescribed to treat infectious diseases have potent antimicrobial activity at physiological concentrations. This anti-bacterial activity raises concern that long-term administration of such nonantibiotics might unintentionally select for multidrug resistance, including resistance to antibiotics. Using Escherichia coli, we show that in most cases, these nonantibiotics do not broadly select for resistance to antibiotics in vitro. However, we identified five nonantibiotics that repeatedly selected for resistance to multiple antibiotics through a shared mechanism of action-upregulation of the multidrug efflux pump AcrAB-TolC. These findings highlight that while the overall risk is low, certain nonantibiotics may still contribute to the emergence of multidrug resistance. Identifying these high-risk drugs can help guide safer prescribing practices and inform strategies to limit the spread of antibiotic resistance.

Escherichia coli↗

PubMed2004

Environmental genome shotgun sequencing of the Sargasso Sea.

We have applied "whole-genome shotgun sequencing" to microbial populations collected en masse on tangential flow and impact filters from seawater samples collected from the Sargasso Sea near Bermuda. A total of 1.045 billion base pairs of nonredundant sequence was generated, annotated, and analyzed to elucidate the gene content, diversity, and relative abundance of the organisms within these environmental samples. These data are estimated to derive from at least 1800 genomic species based on sequence relatedness, including 148 previously unknown bacterial phylotypes. We have identified over 1.2 million previously unknown genes represented in these samples, including more than 782 new rhodopsin-like photoreceptors. Variation in species present and stoichiometry suggests substantial oceanic microbial diversity.

Archaea↗

PubMed1997

[The bacteriology of tuberculosis and non-tuberculosis mycobacterial infections].

Changing incidence and nature of mycobacterial infections subsequent to the historical regression of tuberculosis and the acquired human immunodeficiency syndrome (AIDS) epidemic, as well as the development of new technical tools for molecular biology, have profoundly modified the methods used for the bacteriological diagnosis of mycobacteria infections. Although microscopic search for acid-fast bacilli, culture and antibiotic resistance tests on Löwenstein-Jensen medium remain the reference methods, more rapid and sophisticated methods are now available. Culture on radiolabeled media using the Bactec system has shortened the delay for positive culture and interpretable antibiotic sensitivity tests. Molecular techniques allow: 1) rapid identification of the most frequently isolated mycobacteria strains, including the most frequent laboratory contaminant M. gordonae, with genome probes; 2) genome typing of M. tuberculosis strains to trace interhuman transmission, detect recurrence or exogenous reinfection or demonstrate laboratory contamination; 3) rapid detection of rifampicin resistance; and 4) direct detection of M. tuberculosis and M. avium in pathological specimens. The role of mycobacteria in the environment causing opportunistic infections, atypical mycobacteria or non-tuberculosis mycobacteria (NTM), particularly the aviaire complex, has grown considerably. Isolation and identification relies on methods used to detect bacilli as well as blood cultures and analysis of fecal matter. NTM are naturally resistant to most of the antituberculosis antibiotics but are sometimes sensitive to aminoglycosides, fluoroquinolones or new macrolides.

AIDS-Related Opportunistic Infections↗

PubMed2002

Microbial aldo-keto reductases.

The aldo-keto reductases (AKR) are a superfamily of enzymes with diverse functions in the reduction of aldehydes and ketones. AKR enzymes are found in a wide range of microorganisms, and many open reading frames encoding related putative enzymes have been identified through genome sequencing projects. Established microbial members of the superfamily include the xylose reductases, 2,5-diketo-D-gluconic acid reductases and beta-keto ester reductases. The AKR enzymes share a common (alpha/beta)(8) structure, and conserved catalytic mechanism, although there is considerable variation in the substrate-binding pocket. The physiological function of many of these enzymes is unknown, but a variety of methods including gene disruptions, heterologous expression systems and expression profiling are being employed to deduce the roles of these enzymes in cell metabolism. Several microbial AKR are already being exploited in biotransformation reactions and there is potential for other novel members of this important superfamily to be identified, studied and utilized in this way.

Alcohol Oxidoreductases↗

PubMed2004

zPicture: dynamic alignment and visualization tool for analyzing conservation profiles.

Comparative sequence analysis has evolved as an essential technique for identifying functional coding and noncoding elements conserved throughout evolution. Here, we introduce zPicture, an interactive Web-based sequence alignment and visualization tool for dynamically generating conservation profiles and identifying evolutionarily conserved regions (ECRs). zPicture is highly flexible, because critical parameters can be modified interactively, allowing users to differentially predict ECRs in comparisons of sequences of different phylogenetic distances and evolutionary rates. We demonstrate the application of this module to identify a known regulatory element in the HOXD locus, in which functional ECRs are difficult to discern against the highly conserved genomic background. zPicture also facilitates transcription factor binding-site analysis via the rVista tool portal. We present an example of the HBB complex when zPicture/rVista combination specifically pinpoints to two ECRs containing GATA-1, NF-E2, and TAL1/E47 binding sites that were identified previously as transcriptional enhancers. In addition, zPicture is linked to the UCSC Genome Browser, allowing users to automatically extract sequences and gene annotations for any recorded locus. Finally, we describe how this tool can be efficiently applied to the analysis of nonvertebrate genomes, including those of microbial organisms.

Animals↗

PubMed1997

Experimental surgery to create subgenomes of Bacillus subtilis 168.

The 4,188-kb circular genome of Bacillus subtilis 168 was artificially dissected into two stable circular chromosomes in vivo, one being the 3,878-kb main genome and the other the 310-kb subgenome that was recovered as covalently closed circular DNA in CsCl-ethidium bromide ultracentrifugation. The minimal requirements to physically separate the 310-kb DNA segment out of the genome were two interrepeat homologous sequences and an origin of DNA replication between them. The subgenome originated from the 1,255-1, 551-kb region of the B. subtilis genome was essential for the cell to survive because the subgenome was not lost from the cell. The finding that the B. subtilis genome has a potential to be divided and the resulting two replicons stably maintained may shed light on origins and formation mechanisms of giant plasmids or second chromosomes present in many bacteria. Similar excision or its reversal process, i.e., integration of large sized covalently closed circular DNA pieces into the main genome, implies significant roles of subgenomes in the exchange of genetic information and size variation of bacterial genomes in bacterial evolution.

Bacillus subtilis↗

PubMed2005

Metagenomic analysis of the microbial community at Zodletone Spring (Oklahoma): insights into the genome of a member of the novel candidate division OD1.

A metagenomic library was constructed from the anaerobic sediments of a mesophilic sulfur spring. Thirty-five bacterial 16S rRNA gene-containing clones were identified in this library. Analysis of a genomic fragment belonging to candidate division OD1 provided useful insights into the physiology and biochemistry of this novel, yet-uncultured candidate division.

Bacteria, Anaerobic↗

PubMed2000

Molecular epidemiology of Pseudomonas aeruginosa from cystic fibrosis in Sicily: genome macrorestriction analysis and rapid PCR-ribotyping.

This study addresses the epidemiologic relatedness of a collection of Pseudomonas aeruginosa isolates from cystic fibrosis patients attending the Pediatric Clinic, Catania, Sicily. Genome macrorestriction analysis after pulsed field gel electrophoresis (PFGE) was used to characterise all strains. Furthermore, a rapid typing procedure, developed in this study, based on polymerase chain reaction amplified ribosomal DNA spacer polymorphisms (PCR-ribotyping), straight from bacterial cultures, was used. On the basis of macrorestriction analysis after PFGE, persistence of infection was shown in all patients; two cross-transmission episodes were identified in the nosocomial as well as in the familiar environment. PCR-ribotyping proved to be useful for a DNA-based identification test, suitable for screening purposes. The rapid amplification protocol here tested is proposed to evaluate the discriminatory power of other specific target sequences in PCR-based typing assays, for epidemiologic purposes.

Adolescent↗

PubMed2024

Investigating the degradation potential of microbial consortia for perfluorooctane sulfonate through a functional "top-down" screening approach.

Perfluorooctane sulfonate (PFOS) is a prominent perfluorinated compound commonly found in the environment, known to pose various risks to human health. However, the removal of PFOS presents significant challenges, primarily due to the limited discovery of bacteria capable of effectively degrading PFOS. Moreover, single degradation bacteria often encounter obstacles in individual cultivation and the breakdown of complex pollutants. In contrast, microbial consortia have shown promise in pollutant degradation. This study employed a continuous enrichment method, combined with multiple co-metabolic substrates, to investigate a microbial consortium with the potential for PFOS degradation. By employing this methodology, we effectively identified a microbial consortium that demonstrated the capacity to reduce PFOS when exposed to an optimal concentration of methanol. The consortium predominantly comprised of Hyphomicrobium species (46.7%) along with unclassified microorganisms (53.0%). Over a duration of 20 days, the PFOS concentration exhibited a notable decrease of 56.7% in comparison to the initial level, while considering the exclusion of adsorption effects. Furthermore, by comparing the predicted metabolic pathways of the microbial consortium with the genome of a known chloromethane-degrading bacterium, Hyphomicrobium sp. MC1, using the KEGG database, we observed distinct variations in the metabolic pathways, suggesting the potential role of the unclassified microorganisms. These findings underscore the potential effectiveness of a "top-down" functional microbial screening approach in the degradation of stubborn pollutants.

Fluorocarbons↗

PubMed2001

Cross-resistance testing of antihepadnaviral compounds using novel recombinant baculoviruses which encode drug-resistant strains of hepatitis B virus.

Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains.

2-Aminopurine↗

PubMed2004

TETRA: a web-service and a stand-alone program for the analysis and comparison of tetranucleotide usage patterns in DNA sequences.

BACKGROUND: In the emerging field of environmental genomics, direct cloning and sequencing of genomic fragments from complex microbial communities has proven to be a valuable source of new enzymes, expanding the knowledge of basic biological processes. The central problem of this so called metagenome-approach is that the cloned fragments often lack suitable phylogenetic marker genes, rendering the identification of clones that are likely to originate from the same genome difficult or impossible. In such cases, the analysis of intrinsic DNA-signatures like tetranucleotide frequencies can provide valuable hints on fragment affiliation. With this application in mind, the TETRA web-service and the TETRA stand-alone program have been developed, both of which automate the task of comparative tetranucleotide frequency analysis. AVAILABILITY: http://www.megx.net/tetra. RESULTS: TETRA provides a statistical analysis of tetranucleotide usage patterns in genomic fragments, either via a web-service or a stand-alone program. With respect to discriminatory power, such an analysis outperforms the assignment of genomic fragments based on the (G+C)-content, which is a widely-used sequence-based measure for assessing fragment relatedness. While the web-service is restricted to the calculation of correlation coefficients between tetranucleotide usage patterns of submitted DNA sequences, the stand-alone program generates a much more detailed output, comprising all raw data and graphical plots. The stand-alone program is controlled via a graphical user interface and can batch-process a multitude of sequences. Furthermore, it comes with pre-computed tetranucleotide usage patterns for 166 prokaryote chromosomes, providing a useful reference dataset and source for data-mining. CONCLUSIONS: Up to now, the analysis of skewed oligonucleotide distributions within DNA sequences is not a commonly used tool within metagenomics. With the TETRA web-service and stand-alone program, the method is now accessible in an easy to use manner for a broad audience. This will hopefully facilitate the interrelation of genomic fragments from metagenome libraries, ultimately leading to new insights into the genetic potentials of yet uncultured microorganisms.

Base Composition↗

PubMed2000

Heteroduplex resolution using T7 endonuclease I in microbial community analyses.

Microbial community analyses using molecular techniques, such as PCR followed by genomic library construction, have been helpful in better understanding microbial communities. This is especially critical in ecological systems where most of the microbes present cannot be cultured using traditional techniques. Unfortunately, there are problems associated with the use of such molecular techniques for the analysis of microbial community structure, primarily from the frequent formation of PCR artifacts. Multitemplate PCR is often subject to errors such as heteroduplex formation that is generated during the amplification of a particular gene from a mixed community of DNA. Based on work in this laboratory, heteroduplexes may be resolved before carrying out genomic library construction by including a digestion step with T7 endonuclease I. Here, the 18S rDNA gene of fungi was amplified from soil community DNA and digested with T7 endonuclease I to resolve any heteroduplexes present in the PCR product before cloning. These samples were compared with replicates that did not receive the T7 endonuclease I treatment. Digestion of the amplified community 18S rDNA with 10 U T7 endonuclease I/microgram DNA prior to cloning eliminated heteroduplexes, leaving only the desired clones. Without the T7 endonuclease I treatment, heteroduplexes were produced in approximately 10% of the recombinants screened. The addition of this step may eliminate heteroduplexes from PCR products and ensure that subsequent genomic library construction is not compromised.

Artifacts↗

PubMed2002

Whole genome amplification--applications and advances.

The concept of whole genome amplification is something that has arisen in the past few years as the polymerase chain reaction (PCR) has been adapted to replicate regions of genomes that are of biological interest. The applications are many--forensic science, embryonic disease diagnosis, bioterrorism genome detection, "immortalization" of clinical samples, microbial diversity, and genotyping. Several recent papers suggest that whole genomes can be replicated without bias or non-random distribution of the target, these findings open up a new avenue to molecular biology.

Animals↗

PubMed2000

Signature-tagged mutagenesis in the identification of virulence genes in pathogens.

Signature-tagged mutagenesis is a functional genomics technique that identifies microbial genes required for infection within an animal host, or within host cells. The application of this technique to a range of microbial pathogens has resulted in the identification of novel virulence determinants in each screen performed to date, so that cumulatively several hundred genes have been ascribed a role in virulence.

Candida↗

PubMed2000

Conjugation and transformation of Streptomyces species by tylosin resistance.

The tlrB gene from Streptomyces fradiae has been cloned and used to construct bifunctional Streptomyces-Escherichia coli shuttle vectors carrying the antibiotic resistance genes to kanamycin-neomycin, thiostrepton and tylosin as selection markers. In the same way, the tlrB gene was subcloned in plasmids including the apramycin resistance gene and the oriT sequence from the plasmid pSET152 to facilitate conjugation of Streptomyces spores. The usefulness of the tlrB gene as tylosin resistance marker was ascertained in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor, but not in Streptomyces clavuligerus. The tlrB gene constitutes a useful selection marker when high-frequency of conjugation/transformation is not required or as secondary marker in recombinant Streptomyces species where thiostrepton and kanamycin have been utilized for primary selection.

Bacterial Proteins↗