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Genomic and mechanistic insights into the biodegradation of organic pollutants.

Several new methodologies have enabled recent studies on the microbial biodegradation mechanisms of organic pollutants. Culture-independent techniques for analysis of the genetic and metabolic potential of natural and model microbial communities that degrade organic pollutants have identified new metabolic pathways and enzymes for aerobic and anaerobic degradation. Furthermore, structural studies of the enzymes involved have revealed the specificities and activities of key catabolic enzymes, such as dioxygenases. Genome sequencing of several biodegradation-relevant microorganisms have provided the first whole-genome insights into the genetic background of the metabolic capability and biodegradation versatility of these organisms. Systems biology approaches are still in their infancy, but are becoming increasingly helpful to unravel, predict and quantify metabolic abilities within particular organisms or microbial consortia.

Bacteria↗

Genome streamlining in a cosmopolitan oceanic bacterium.

The SAR11 clade consists of very small, heterotrophic marine alpha-proteobacteria that are found throughout the oceans, where they account for about 25% of all microbial cells. Pelagibacter ubique, the first cultured member of this clade, has the smallest genome and encodes the smallest number of predicted open reading frames known for a free-living microorganism. In contrast to parasitic bacteria and archaea with small genomes, P. ubique has complete biosynthetic pathways for all 20 amino acids and all but a few cofactors. P. ubique has no pseudogenes, introns, transposons, extrachromosomal elements, or inteins; few paralogs; and the shortest intergenic spacers yet observed for any cell.

Alphaproteobacteria↗

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Base Sequence↗

Insights into the genomes of archaea mediating the anaerobic oxidation of methane.

The anaerobic oxidation of methane is a globally significant process which is mediated by consortia of yet uncultivated methanotrophic archaea (ANME) and sulfate-reducing bacteria. In order to gain deeper insights into genome characteristics of the different ANME groups, large-insert genomic libraries were constructed using DNA extracted from a methanotrophic microbial mat growing in the anoxic part of the Black Sea, and from sediments above gas hydrates at the Hydrate Ridge off the coast of Oregon. Analysis of these fosmid libraries with respect to archaeal 16S rRNA gene diversity revealed a single ANME-1b ribotype for the Black Sea libraries, whereas the sequences derived from the Hydrate Ridge library phylogenetically affiliated with the ANME-2a, ANME-2c and ANME-3 group. Genome walking for ANME-1b resulted in a contiguous 155 kb composite genome fragment. The comparison of a set of four genomic fragments belonging to the different ANME groups revealed differences in the rRNA operon structure and the average G+C content, with the ANME-2c contig showing the highest divergence within the set. A detailed analysis of the ANME contigs with respect to genes putatively involved in the anaerobic oxidation of methane led to the identification of: (i) a putative N5,N10-methenyltetrahydromethanopterin cyclohydrolase gene, (ii) a gene cluster supposedly encoding a novel type of heterodisulfide reductase/dehydrogenase complex and (iii) a gene cluster putatively encoding a new type of CO dehydrogenase/acetyl-CoA synthase enzyme complex.

Amino Acid Sequence↗

The application of AI-driven and engineered intratumoral microbes in cancer therapy.

BACKGROUND: Although investigations of the intratumoral microbiota date back thousands of years, breakthrough transformations have only recently been achieved through high-throughput sequencing and multiomic technologies. These advances have revealed diverse and tumor type-specific microbial communities that drive carcinogenesis via immunomodulation, metabolic reprogramming, and genomic instability. Current cornerstones of cancer therapies-including chemotherapy, radiotherapy, immunotherapy, and targeted therapy-are limited by systemic toxicity, localized tissue damage, drug resistance, and low patient response rates. These constraints underscore the urgent need for more effective and precise therapeutic strategies. MAIN BODY: This review comprehensively integrates artificial intelligence (AI) technologies into the characterization of the intratumoral microbiota, facilitating the development of novel computational pipelines for mapping microbe-host crosstalk. We systematically summarize recent advances in engineered microbial therapeutics, including bacteria designed for targeted antitumor activity and engineered microorganisms that enable the localized delivery of therapeutic agents. Furthermore, this review critically evaluates the safety profiles of microbiota-based interventions and discusses key challenges in clinical translation. CONCLUSIONS: By combining cutting-edge computational technologies, biological research, and clinical insights, this review aims to bridge the gap between microbiome science and oncological practice, pioneering innovative strategies for microbiota-guided diagnostics and personalized cancer therapy.

Humans↗

Yeast genomic expression studies using DNA microarrays.

The exploration and characterization of yeast genomic expression programs is providing a wealth of information about yeast biology, as well as other organisms. The intriguing biology of yeast species invites characterization of genomic expression patterns to illuminate the details of cellular physiology. In addition to its value as an interesting organism, yeast maintains its role as an excellent model in which to characterize genomic expression programs. Microarray studies are quickly spreading to plant, animal, and microbial organisms that remain in the early stages of characterization. The extensive knowledge of yeast biology, as well as the relative ease with which yeast studies can be performed and controlled, facilitates interpretation of the genomic expression data. Importantly, existing information about yeast biology, including functional annotations for each gene, is captured and efficiently presented in databases such as the Saccharomyces Genome Database (SGD), the Munich Information Center Yeast Genome Database (MIPS), the Yeast and Pombe Protein Databases (YPD and PPD, respectively), and others. A number of databases also allow the exploration of published genomic expression studies, including the "Expression Connection" at SGD and the Microarray Global Viewer (yMGV) organized by Marc et al. Consulting these databases to retrieve known details about gene function and regulation vastly facilitates interpretation of the genomic expression data, allowing biological hypotheses to be formulated and tested. These hypotheses can be applied to other organisms that may execute genomic expression programs similar to those seen in yeast. Furthermore, as more genomic expression studies in multiple organisms emerge, large-scale data comparisons can be conducted, within and across organisms. Incorporating the results of yeast studies into such comparisons is certain to increase our understanding about the function, regulation, and evolution of genomic expression programs.

Carbocyanines↗

Gene fusion in Helicobacter pylori: making the ends meet.

Fusion genes have been reported as a means of enabling the development of novel or enhanced functions. In this report, we analyzed fusion genes in the genomes of two Helicobacter pylori strains (26695 and J99) and identified 32 fusion genes that are present as neighbours in one strain (components) and are fused in the second (composite), and vice-versa. The mechanism for each case of gene fusion is explored. 28 out of 32 genes identified as fusion products in this analysis were reported as essential genes in the previously documented transposon mutagenesis of H. pylori strain G27. This observation suggests the potential of the products of fusion genes as putative microbial drug targets. These results underscore the utility of bacterial genomic sequence comparisons for understanding gene evolution and for in silico drug target identification in the post-genomic era.

Amino Acid Sequence↗

Crawling under the radar: Two novel Paulinella species expand knowledge about the ecology and evolution of a primary plastid-containing amoeba lineage.

The genus Paulinella represents a rare, independent case of primary endosymbiosis, providing a unique system to study the early stages of organelle evolution. Here, we expand current understanding of primary plastid endosymbiosis through the discovery and characterization of two novel photosynthetic amoebae, Paulinella marae sp. nov. and Paulinella murrayi sp. nov., isolated from a brackish water habitat in North Carolina, United States. Complete chromatophore genomes and mitochondrial data revealed conserved gene content but notable structural variation, including genome rearrangements and inversion events. Phylogenetic analyses uncovered significant discordance between nuclear and organelle datasets, likely driven by substitution saturation, limited taxon sampling, and differing evolutionary signals across loci. Ecological observations over multiple years indicate that both species are in low abundance but consistently present, and when coupled with hobbyist data, support the hypothesis that photosynthetic Paulinella species are globally distributed yet under-sampled. These results increase known species diversity within the clade from four to six and highlight the importance of integrating field-based observations with genomic approaches. Overall, this work advances Paulinella as a model for studying ongoing primary endosymbiosis, lineage divergence, and the ecological strategies of low-abundance microbial eukaryotes.

Paulinella↗

Sequence-based analysis of metabolic demands for protein synthesis in prokaryotes.

Constraints-based models for microbial metabolism can currently be constructed on a genome-scale. These models do not account for RNA and protein synthesis. A scalable formalism to describe translation and transcription that can be integrated with the existing metabolic models is thus needed. Here, we developed such a formalism. The fundamental protein synthesis network described by this formalism was analysed via extreme pathway and flux balance analyses. The protein synthesis network exhibited one extreme pathway per messenger RNA synthesized and one extreme pathway per protein synthesized. The key parameters in this network included promoter strengths, messenger RNA half-lives, and the availability of nucleotide triphosphates, amino acids, RNA polymerase, and active ribosomes. Given these parameters, we were able to calculate a cell's material and energy expenditures for protein synthesis using a flux balance approach. The framework provided herein can subsequently be integrated with genome-scale metabolic models, providing a sequence-based accounting of the metabolic demands resulting from RNA and protein polymerization.

Amino Acids↗

Histidine biosynthesis in plants.

The study of histidine metabolism has never been at the forefront of interest in plant systems despite the significant role that the analysis of this pathway has played in development of the field of molecular genetics in microbes. With the advent of methods to analyze plant gene function by complementation of microbial auxotrophic mutants and the complete analysis of plant genome sequences, strides have been made in deciphering the histidine pathway in plants. The studies point to a complex evolutionary origin of genes for histidine biosynthesis. Gene regulation studies have indicated novel regulatory networks involving histidine. In addition, physiological studies have indicated novel functions for histidine in plants as chelators and transporters of metal ions. Recent investigations have revealed intriguing connections of histidine in plant reproduction. The exciting new information suggests that the study of plant histidine biosynthesis has finally begun to flower.

Arabidopsis↗

Checkerboard DNA-DNA hybridisation technology focused on the analysis of Gram-positive cariogenic bacteria.

Checkerboard DNA-DNA hybridisation enabled the quantitative analysis of plaque samples against 40 microbial species simultaneously, using digoxygenin-labelled, whole-genome DNA probes. This technique was initially developed to study the predominantly Gram-negative sub-gingival plaque microbiota. The aim of this study was to apply it to a suite of predominantly Gram-positive microorganisms, such as those implicated in cariogenesis. To specifically target Gram-positive species (and Candida albicans) required optimisation and modification of DNA extraction, prehybridisation, hybridisation, and antibody detection conditions. The suitability of the revised technique for clinical and epidemiological studies was confirmed using interproximal plaque from small groups of 5- to 6-year-old children of high (decayed, missing, or filled teeth (dmft)> or =5, n=8) and zero (n=5) caries rates.

Buffers↗

Honor thy symbionts.

Our intestine is the site of an extraordinarily complex and dynamic environmentally transmitted consortial symbiosis. The molecular foundations of beneficial symbiotic host-bacterial relationships in the gut are being revealed in part from studies of simplified models of this ecosystem, where germ-free mice are colonized with specified members of the microbial community, and in part from comparisons of the genomes of members of the intestinal microbiota. The results emphasize the contributions of symbionts to postnatal gut development and host physiology, as well as the remarkable strategies these microorganisms have evolved to sustain their alliances. These points are illustrated by the human-Bacteroides thetaiotaomicron symbiosis. Interdisciplinary studies of the effects of the intestinal environment on genome structure and function should provide important new insights about how microbes and humans have coevolved mutually beneficial relationships and new perspectives about the foundations of our health.

Animals↗

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proteomics: a new era in anaerobic microbiology.

Genome sequence data provide a framework for predicting potential microbial activities; however, the proteome content of the cell dictates its response to its environment. Microbiology is witnessing a major initiative to elucidate the nature of the proteome of large numbers of species. The tool driving the proteomic revolution is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. During the analysis process, proteins are ionized and separated on the basis of their mass-to-charge ratios, which results in a characteristic mass-spectral profile. Because of the dynamic nature of the cell and the large number of external parameters that could influence its mass-spectral profile, considerable work was needed initially to optimize sample analysis and obtain consistent and reproducible results. For many anaerobes that grow poorly or are nonreactive in most diagnostic systems, proteome analysis is likely to have a major impact on microbial diagnosis and the delineation of centers of diversity associated with infections.

Bacteria, Anaerobic↗

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi.

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Amino Acid Sequence↗

SdrX, a serine-aspartate repeat protein expressed by Staphylococcus capitis with collagen VI binding activity.

Staphylococcus capitis (S. capitis) has been implicated in a large proportion of coagulase-negative staphylococcal infections in very-low-birth-weight infants. To identify potential therapeutic targets, the S. capitis genome was probed for the presence of genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM). By using Southern blot analysis, an S. capitis gene, designated sdrX, that contained sequence motifs consistent with the Sdr family of MSCRAMM proteins was identified. By using monospecific antisera in Western blot and flow cytometry, SdrX was demonstrated to be expressed on the surface of S. capitis. Human collagen type VI was found to bind both the recombinant A domain of SdrX and viable S. capitis expressing SdrX. SdrX is the first collagen-binding Sdr protein described and is the first MSCRAMM protein identified in S. capitis.

Adhesins, Bacterial↗

Beyond borders: plasmids drive a shared antibiotic resistome in European urban water systems.

BACKGROUND: Urban wastewater systems (UWSs) act as reservoirs and conduits for the dissemination of antibiotic resistance genes (ARGs), with plasmids playing a central role in their spread. Despite their significance, the diversity and persistence of plasmids in UWSs remain underexplored. RESULTS: This study applies a multi-omics approach, including metagenomic and direct plasmidome sequencing, high-throughput qPCR array, and whole genome sequencing of plasmid isolates, to comprehensively profile the microbial plasmidome and resistome on 78 samples across UWSs in Denmark, Spain, and the UK. We successfully uncovered an extensive plasmid and ARG diversity that could not be fully captured by a single method, especially identified 78,574 plasmids, including 20,925 plasmids previously unreported. We also observed that plasmids carried a disproportionate share of clinically relevant ARGs, particularly beta-lactamase resistance genes; most importantly, they were preferentially located on transmissible plasmids. Furtherly, plasmids harbor ARG can enhance their persistence in wastewater ecosystems, especially harboring multiple types of ARGs. Moreover, Bacteroides emerged as a unique persistent ARG reservoir not only for harboring and disseminating diverse resistance genes especially in residential-relevant areas, but also emerged as a major driver of antimicrobial resistance dynamics across different wastewater treatment processes. CONCLUSIONS: Overall, this work provides the first attempt at a holistic description of the UWSs' resistome, its structure, dynamics, and mobility and significantly expands the current knowledge. Video Abstract.

Plasmids↗

Defects in pyruvate kinase cause a conditional increase of thiamine synthesis in Salmonella typhimurium.

As genomic sequence data become more prevalent, the challenges in microbial physiology shift from identifying biochemical pathways to understanding the interactions that occur between them to create a robust but responsive metabolism. One of the most powerful methods to identify such interactions is in vivo phenotypic analysis. We have utilized thiamine synthesis as a model to detect subtle metabolic interactions due to the sensitivity allowed by the small cellular requirement for this vitamin. Although purine biosynthesis produces an intermediate in thiamine synthesis, mutants blocked in the first step of de novo purine biosynthesis (PurF) are able to grow in the absence of thiamine owing to an alternative synthesis. A number of general metabolic defects have been found to prevent PurF-independent thiamine synthesis. Here we report stimulation of thiamine-independent growth caused by a mutation in one or both genes encoding the pyruvate kinase isozymes. The results presented herein represent the first phenotype described for mutants defective in pykA or pykF, and thus identify metabolic interactions that exist in vivo.

Gene Expression Regulation, Bacterial↗

Polymerase chain reaction from bench to bedside. Applications for infectious disease.

The past decade has seen the rapid advancement of molecular biology and its application in the field of infectious diseases. The polymerase chain reaction (PCR) is a technique which allows for the in vitro amplification of DNA. The ability of this method to recognize and amplify small quantities of foreign genome from unpurified samples will revolutionize the diagnoses of microbial diseases which have previously been cumbersome or impossible. It has already been widely applied in this area, and clinical labs will soon be offering this test for selected pathogens. There are ongoing studies evaluating its use in CNS infections and for the identification of viruses in the immunocompromised host. The PCR is not without problems, particularly false positive results from contamination. Because it is so sensitive, the significance of a positive result in patients with latent or chronic infections is yet to be determined. As our knowledge and familiarity with PCR expands, it will become a powerful tool for the clinician to use to identify various infectious diseases.

Communicable Diseases↗