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PubMed1999

The pimB gene of Mycobacterium tuberculosis encodes a mannosyltransferase involved in lipoarabinomannan biosynthesis.

The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosis genomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.

Bacterial Proteins↗
PubMedDate not supplied

Super-integrons.

Integrons represent the primary mechanism for antibiotic resistance gene capture and dissemination among gram-negative bacteria. The recent finding of super-integron (SI) structures in the genomes of several bacterial species has expanded their role in genome evolution. The Vibrio cholerae superintegron is gathered in a single chromosomal super-structure harbouring hundreds of gene cassettes. The encoded functions, when identifiable, are linked to adaptations extending beyond antibiotic resistance and pathogenicity. Comparison of the cassette contents of super-integrons from remote Vibrio species suggests that most of their cassettes are species-specific. Many bacterial species belonging to several distinct genera of the gamma- and beta-proteobacteria undoubtedly carry or show strong evidence for the presence of chromosomal SIs. If each bacterial species harbouring a SI has its own cassette pool, the resource in terms of gene cassette availability may be immense.

Base Sequence↗
PubMed2026

Genome-based predictions of metabolic preferences and substrate phenotypes in psychrotrophic bacteria from permafrost environments.

Genomes reveal vast functional potential, but harbor genomic noise that obscures prediction of metabolic and environmental preferences. Genomic databases are skewed towards clinically relevant and easily cultivated bacteria, limiting predictions for diverse and underrepresented environmental taxa. Psychrotrophic bacteria, which can survive and grow in cold, nutrient-limited, dry, and saline environments, are especially underrepresented despite their relevance for understanding microbial responses to changing cold environments and potential biotechnological value given growth at low temperatures. Assembling complete genomes of 48 isolates from Alaskan permafrost, seasonally frozen active layer soils, and terrestrial ice, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog annotations to evaluate the predictability of metabolic resource-use traits observed using phenotypic tests. Genome-predicted values for glycolytic versus gluconeogenic catabolic preference index, or sugar-acid preference (SAP), explained over 50% of the variance in empirically observed SAP. SAP was inversely correlated to genomic GC content, which follows phylum-level trends, indicating that coarse metabolic preference covaries with phylogeny. Regularized elastic net models offered a more granular view, linking KEGG genes to specific substrate utilization and sensitivity phenotypes and yielding moderate but reproducible accuracy (AUC 0.70-0.79) for 11 substrates, demonstrating that specific substrate responses may be predictable from relatively small subsets of KO genes. These results extend recent advances, such as the SAP metric, and highlight associations among genomic GC content, phylum, and broad metabolic strategy. Linking genomic content to phenotype using isolates is a necessary step toward predictive models of microbial function in environmental communities, and this work can be used for hypothesis generation, with applications towards more expansive data sets.IMPORTANCECold region soils and ice host psychrotrophic bacteria with metabolic traits and adaptations that enable persistence in harsh, resource-limited environments. However, these taxa are underrepresented in genomic reference databases dominated by well-studied, mesophilic organisms. This gap limits inference of ecological strategies and our ability to predict how these microbes may influence the large, thaw-vulnerable carbon reservoirs in permafrost. Here, we show that genomic GC content is associated with the sugar-versus-acid catabolic preference (SAP) of isolates across major phyla, suggesting that broad genomic features may provide a coarse signal of metabolic strategy. We demonstrate that a modified SAP metric, using binary (positive/negative) substrate utilization rather than detailed growth rate measurements, is moderately predictive, thus extending its application to slow-growing or difficult-to-culture taxa. Together, these advances broaden the toolkit for linking genome content to resource-use traits (phenotype) in poorly characterized, cold-adapted bacteria and offer a tractable entry point to broad prediction and hypothesis generation.

Genome, Bacterial↗
PubMed2001

Enterococcus faecalis multi-drug resistance transporters: application for antibiotic discovery.

Using bioinformatics approaches, 34 potential multidrug resistance (MDR) transporter sequences representing 4 different transporter families were identified in the unannotated Enterococcus faecalis database (TIGR). A functional genomics campaign generating single-gene insertional disruptions revealed several genes whose absence confers significant hypersensitivities to known antimicrobials. We constructed specific strains, disrupted in a variety of previously unpublished, putative MDR transporter genes, as tools to improve the success of whole-cell antimicrobial screening and discovery. Each of the potential transporters was inactivated at the gene level and then phenotypically characterized, both with single disruption mutants and with 2-gene mutants built upon a delta norA deleted strain background.

ATP-Binding Cassette Transporters↗
PubMed2026

Human skin microbiota and postpartum depression: A bidirectional Mendelian randomization study.

Postpartum depression (PPD) is a common mental health disorder after childbirth. Although microbiome research in PPD has mainly focused on the gut, the role of skin microbiota remains unclear. We used Mendelian randomization (MR) to assess potential causal associations between skin microbiota and PPD. A bidirectional 2-sample MR analysis used genome-wide association study (GWAS) summary statistics. Genetic instruments for skin microbial features were obtained from a published skin microbiota GWAS, and PPD data were derived from 67,205 mothers (7604 cases, 59,601 controls). Instruments were selected at P&#x2005;<1&#x2005;&#xd7;&#x2005;10-5, linkage disequilibrium-clumped, harmonized, and filtered for weak instruments (F statistic&#x2005;<10). Because this microbiome threshold is exploratory, Benjamini-Hochberg false discovery rate correction was applied within taxonomic levels. The inverse-variance weighted method was primary, complemented by weighted median and mode-based methods. Heterogeneity, pleiotropy, and outliers were assessed using Cochran Q, MR-Egger intercept, and MR-PRESSO. Three skin microbial taxa showed nominal associations with PPD. Higher genetically predicted Acinetobacter on the dorsal forearm (dry skin; 9 single nucleotide polymorphisms [SNPs]; mean F&#x2005;=&#x2005;22.12) and Proteobacteria in the antecubital fossa (moist skin; 6 SNPs; mean F&#x2005;=&#x2005;23.44) were associated with increased PPD risk, whereas Betaproteobacteria in the antecubital fossa (11 SNPs; mean F&#x2005;=&#x2005;21.54) was associated with decreased risk. Associations were directionally consistent, with no substantial heterogeneity or horizontal pleiotropy. After multiple-testing assessment, the findings were exploratory rather than definitive. Reverse MR did not support an effect of PPD on the identified skin microbiota. This MR study provides exploratory genetic evidence linking specific skin microbial features to PPD risk. The findings extend microbiota-related hypotheses beyond the gut microbiome but require validation in larger microbiome GWAS datasets, longitudinal cohorts, and mechanistic studies before clinical or causal conclusions are drawn.

Humans↗
PubMed1998

Mapping the germ-line and somatic genomes of a ciliated protozoan, Tetrahymena thermophila.

Ciliates are among the very few eukaryotes in which the powers of molecular biology, conventional genetics, and microbial methodology can be synergistically combined. Because ciliates also are distant relatives of vertebrates, fungi, and plants, the sequencing of a ciliate genome will be of import to our understanding of eukaryotic biology. Tetrahymena thermophila is the only ciliate in which a systematic genetic mapping of DNA polymorphisms has begun. Tetrahymena has many biological features that make it a specially or uniquely useful experimental system for fundamental research in cell and molecular biology and for biotechnological applications. A key factor in the usefulness of Tetrahymena is the speed, facility, and versatility with which it can be cultivated under a wide range of nutrient conditions, temperature, and scale. This article describes the progress made in genetically and physically mapping the genomes of T. thermophila: the micronuclear (germ-line) genome, which is not transcriptionally expressed, and the macronuclear (somatic) fragmented genome, which is actively expressed and determines the cell's phenotype.

Animals↗
PubMed2025

Paratyphoid fever and the genomics of Salmonella enterica serovar Paratyphi A in Taiwan.

BACKGROUND: Salmonella enterica serovar Paratyphi A (S. Paratyphi A) has emerged as a significant global health concern due to the progressive development of antimicrobial resistance and its broader geographic distribution. In Taiwan, paratyphoid fever was historically rare and predominantly associated with imported cases. Since 2022, however, a marked increase in domestically acquired infections has been observed, prompting investigations into their origin and likely route of introduction. METHODS: We analyzed surveillance data on 223 patients with paratyphoid fever reported in Taiwan between January 2001 and December 2024. Whole-genome sequencing and antimicrobial susceptibility testing were performed on 88 S. Paratyphi A isolates obtained from both imported and domestically acquired infections from 2007 to 2024. Phylogenetic analysis and genotyping were conducted to assess genetic relatedness and to trace potential sources of introduction by comparing them with global isolates. RESULTS: Although 55.2% of paratyphoid fever infections were imported, domestically acquired infections became predominant after 2022. Most isolates (76.1%) were resistant to nalidixic acid and nonsusceptible to ciprofloxacin due to gyrA mutations at codon 83 (S83F or S83Y). The majority of domestic isolates were classified as ST129 and paratype 2.4 and showed close genetic relatedness to strains from Indonesia. Of the 31 domestic isolates collected between 2022 and 2024, 30 clustered with Indonesian strains, and 28 exhibited nearly identical genomic profiles, which suggested a prolonged outbreak likely linked to a common external source, such as contaminated imported food. CONCLUSIONS: The genomic evidence suggests that the recent increase in domestically acquired S. Paratyphi A infections in Taiwan represents a prolonged outbreak rather than a sustained epidemiological shift. These infections were closely related to strains from Indonesia, suggesting a potential epidemiological link between the two countries in the transmission of paratyphoid fever. While 76.1% of isolates were nonsusceptible to ciprofloxacin due to gyrA mutations, susceptibility to traditional first-line agents remained high. The observed decline in case numbers in 2024 may indicate that the outbreak is subsiding. Genomic surveillance played a crucial role in tracing sources of infection and informing targeted public health responses.

Paratyphoid Fever↗
PubMed1994

Cloning of the Saccharomyces cerevisiae gene whose overexpression overcomes the effects of HM-1 killer toxin, which inhibits beta-glucan synthesis.

A gene whose overexpression can endow Saccharomyces cerevisiae cells with resistance to HM-1 killer toxin was cloned from an S. cerevisiae genomic library. This gene, designated HKR1 (Hansenula mrakii killer toxin-resistant gene 1), contains a 5.4-kb open reading frame. The predicted amino acid sequence of the protein specified by HKR1 indicates that the protein consists of 1,802 amino acids and is very rich in serine and threonine, which could serve as O-glycosylation sites. The protein also contains two hydrophobic domains at the N-terminal end and in the C-terminal half, which could function as a signal peptide and transmembrane domain, respectively. Hkr1p is found to contain an EF hand motif of the calcium-binding consensus sequence in the C-terminal cytoplasmic domain. Thus, Hkr1p is expected to be a calcium-binding, glycosylated type I membrane protein. Southern and Northern (RNA) analyses demonstrated that there is a single copy of the HKR1 gene in the S. cerevisiae genome, and the transcriptional level of HKR1 is extremely low. Gene disruption followed by tetrad analysis showed that HKR1 is an essential gene. Overexpression of the truncated HKR1 encoding the C-terminal half of Hkr1p made the cells more resistant to HM-1 killer toxin than the full-length HKR1 did, demonstrating that the C-terminal half of Hkr1p is essential for overcoming the effect of HM-1 killer toxin. Furthermore, overexpression of HKR1 increased the beta-glucan content in the cell wall without affecting in vitro beta-glucan synthase activity, suggesting that HKR1 regulates beta-glucan synthesis in vivo.

Amino Acid Sequence↗
PubMed2005

The net of life: reconstructing the microbial phylogenetic network.

It has previously been suggested that the phylogeny of microbial species might be better described as a network containing vertical and horizontal gene transfer (HGT) events. Yet, all phylogenetic reconstructions so far have presented microbial trees rather than networks. Here, we present a first attempt to reconstruct such an evolutionary network, which we term the "net of life". We use available tree reconstruction methods to infer vertical inheritance, and use an ancestral state inference algorithm to map HGT events on the tree. We also describe a weighting scheme used to estimate the number of genes exchanged between pairs of organisms. We demonstrate that vertical inheritance constitutes the bulk of gene transfer on the tree of life. We term the bulk of horizontal gene flow between tree nodes as "vines", and demonstrate that multiple but mostly tiny vines interconnect the tree. Our results strongly suggest that the HGT network is a scale-free graph, a finding with important implications for genome evolution. We propose that genes might propagate extremely rapidly across microbial species through the HGT network, using certain organisms as hubs.

Algorithms↗
PubMed2000

Whole genomic DNA probe for detection of Porphyromonas endodontalis.

The purpose of the present study was to develop a DNA probe for Porphyromonas endodontalis. Pure cultures of P. endodontalis were grown in TYP medium, in an anaerobic chamber. DNA was extracted from the P. endodontalis and labeled using the Genius System by Boehringer Mannheim. The labeled P. endodontalis DNA was used in dot-blot hybridization reactions with homologous (P. endodontalis) and unrelated bacterial samples. To determine specificity, strains of 40 other oral bacterial species (e.g. Porphyromonas gingivalis, Porphyromonas asaccharolytica, and Prevotella intermedia) were spotted and reacted with the P. endodontalis DNA probe. None of the panel of 40 oral bacteria hybridized with the P. endodontalis probe, whereas the blot of the homologous organism showed a strong positive reaction. To determine the sensitivity of the probe, dilutions of a P. endodontalis suspension of known concentration were blotted onto a nylon membrane and reacted with the probe. The results of our investigation indicate that the DNA probe that we have prepared specifically detects only P. endodontalis and can detect at least 3 x 10(4) cells.

Colony Count, Microbial↗
PubMed1998

[Human immunodeficiency virus and AIDS in terms of reverse transcriptase and molecular evolution].

Human immunodeficiency virus type 1 (HIV-1) evolves rapidly in the host. The computer analysis of the HIV-1 genome has shown that the mutation manner is dependent on oligonucleotide sequences (in particular, six bases long); thus HIV-1 adaptively evolves. The six-base-long interaction between template-primer oligonucleotide and the reverse transcriptase (RT) has been revealed by the crystal structure of RT, in vitro termination assay of plymerization, and hydroxyl radical footprint analysis. It has been thought that AIDS is caused by the large numbers of HIV-1 quasispecies yielded by the adaptive and rapid evolution in the host. However, the slow evolution and the high levels of viral RNA in the progressive HIV-1 infected individuals (progressives) were recently reported; in contrast, the adaptive and rapid evolution and the low viral-RNA levels were reported in the non-progressives. This suggests that the physiological environment, e.g. pH and dNTP balance, in which RT works in the progressives is different from that in the non-progressives.

Acquired Immunodeficiency Syndrome↗
PubMed1993

The Neurospora uvs-2 gene encodes a protein which has homology to yeast RAD18, with unique zinc finger motifs.

A clone containing the DNA repair gene uvs-2 of Neurospora crassa was identified from a Neurospora genomic DNA library using the sib-selection method. Transformants were screened for resistance to methyl methane sulfonate (MMS). A DNA fragment that complements the uvs-2 mutation was subcloned by monitoring its ability to transform the uvs-2 mutant to MMS resistance. Deletion analysis of the cloned DNA indicated that the size of the uvs-2 gene is approximately 1.6 kb. The identity of the uvs-2 gene was verified by restriction fragment length polymorphism (RFLP) mapping. The sensitivity of the transformant to three different mutagens was similar to that of the wild-type strain. Nucleotide sequences of genomic DNA and cDNA of the uvs-2 gene indicated that it has an open reading frame (ORF) of 1572 bp with a 69 bp intron in the middle of the sequence. Two transcription initiation sites located around 73 bp and 290 bp upstream of the translation initiation codon were identified by primer extension experiments. Northern analysis revealed that the nature transcript of the uvs-2 gene was about 1.8 kb long. The uvs-2 gene ORF is deduced to encode a polypeptide of 501 amino acids with a molecular mass of 54 kDa. The proposed polypeptide has 25% identity to the RAD18 polypeptide of Saccharomyces cerevisiae and contains two unique zinc finger motifs for nucleic acid binding. Similarities between the phenotypes of the rad18 and uvs-2 mutants suggest that the uvs-2 gene encodes a protein which is involved in postreplication repair, rather than excision repair.

Amino Acid Sequence↗
PubMed2026

Novel Insights into Metagenomic-Assembled Genomes from Layer Chicken Housing Environment.

Culture-independent techniques are playing a major role in exploring unique and novel microbial communities from complex ecosystems, leading to an outstanding impact on our basic understanding of the tree of life. Microbial communities are not extensively studied in layer chicken housing environments, particularly from the point of view of taxa carrying antimicrobial resistance genes, virulence genes and their functional potential. This study aimed to extract metagenomic-assembled genomes (MAGs) from the Illumina short-reads shotgun metagenomics sequenced data that originated from an Alberta poultry barn environment and then to study host tracking of antimicrobial resistance genes (ARGs) and the roles of genes involved in functions related to ammonia production, short-chain fatty acid (SCFA)-related pathways, sulfur metabolism, methane emission, stress and disinfectant-related pathways. A total of 251 high-quality MAGs were extracted, including 249 bacterial and two archaeal genomes from sequencing data of 30 metagenomic sequencing samples comprising 15 air and 15 manure samples collected from 15-layer farms. Interestingly 22 bacterial MAGs were not classified to species levels using GTDB-based classification. ARGs were mainly harbored by the genera Staphylococcus, Alistepes, Romboutsia, and Enterococcus. Bacteroides is a main taxon carrying ARGs in air samples. Ammonia production-related genes were mainly tracked in Staphylococcus, Ruminococcus and Corynebacterium genera. The assimilatory sulfate reduction genes responsible for sulfur metabolism and hydrogenase-related genes responsible for hydrogen cycling were traced from Staphylococcus originated from both air and manure. The current study provides characterizations of MAGs from a poultry housing environment by linking microbial taxa with virulence, resistance, and metabolic functions. The findings emphasize the role of microbiota in shaping gas emissions and AMR, with implications for poultry health and worker's safety and the ultimate aim of sustainable poultry production.

Animals↗
PubMed2001

The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons.

Integrons are genetic elements that acquire and exchange exogenous DNA, known as gene cassettes, by a site-specific recombination mechanism. Characterized gene cassettes consist of a target recombination sequence (attC site) usually associated with a single open reading frame coding for an antibiotic resistance determinant. The affiliation of multiresistant integrons (MRIs), which contain various combinations of antibiotic resistance gene cassettes, with transferable elements underlies the rapid evolution of multidrug resistance among diverse Gram-negative bacteria. Yet the origin of MRIs remains unknown. Recently, a chromosomal super-integron (SI) harboring hundreds of cassettes was identified in the Vibrio cholerae genome. Here, we demonstrate that the activity of its associated integrase is identical to that of the MRI integrase, IntI1. We have also identified equivalent integron superstructures in nine distinct genera throughout the gamma-proteobacterial radiation. Phylogenetic analysis revealed that the evolutionary history of the system paralleled that of the radiation, indicating that integrons are ancient structures. The attC sites of the 63 antibiotic-resistance gene cassettes identified thus far in MRIs are highly variable. Strikingly, one-fifth of these were virtually identical to the highly related yet species-specific attC sites of the SIs described here. Furthermore, antimicrobial resistance homologues were identified among the thousands of genes entrapped by these SIs. Because the gene cassettes of SIs are substrates for MRIs, these data identify SIs as the source of contemporary MRIs and their cassettes. However, our demonstration of the metabolic functions, beyond antibiotic resistance and virulence, of three distinct SI gene cassettes indicates that integrons function as a general gene-capture system for bacterial innovation.

Alteromonas↗
PubMed2003

Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities.

Spotted oligonucleotide microarrays potentially offer a wide scope of applications for microbial ecology, especially as they improve the flexibility of design and the specificity of detection compared to PCR product based microarrays. Sensitivity, however, was expected to be problematic, as studies with the more sensitive PCR-based cDNA microarrays indicate that only genes from populations contributing to more than 5% of the community DNA can be detected. We evaluated several parameters to increase sensitivity and then tested applicability for bacterial functional genomics. The optimal parameters were the use of 5'-C6-amino-modified 70-mers printed on CMT-GAPS II substrates at a 40 micro M concentration combined with the use of Tyramide Signal Amplification labelling. This protocol allowed detection of single copy genes belonging to an organism contributing to 1% or more of the total community. To demonstrate its application, we detected the specific aromatic oxygenase genes in a soil community degrading polychlorinated biphenyls (PCBs). This increase in sensitivity is important if oligonucleotide microarrays are to be used for simultaneous monitoring of a range of functions performed by different microorganisms in the environment.

Bacteria↗