Genomes Of Microorganisms Research Articles

Recent advances in engineering non-native microorganisms for poly(3-hydroxybutyrate) production.

Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer that belongs to a group of polymers called polyhydroxyalkanoates (PHAs). PHB can be synthesized from renewable resources, making it a promising alternative to petroleum-derived plastics. It is also considered non-toxic, biodegradable, and biocompatible, which makes it suitable for various applications in the medicine and biomedicine. Many microorganisms biosynthesize and accumulate PHB naturally. However, recent advancements in metabolic engineering and synthetic biology have allowed scientists to engineer non-native microorganisms to produce PHB. This review comprehensively summarizes all non-native microbial hosts used for PHB biosynthesis and discusses different metabolic engineering approaches used to enhance PHB production. These strategies include optimizing the biosynthesis pathway through cofactor engineering, metabolic pathway reconstruction, and cell morphology engineering. Moreover, the CRISPR/Cas9 approach is also used for manipulating the genome of non-host microorganisms to enable them produce PHB. Among non-native microbial hosts, Escherichia coli has been successfully used for industrial-scale PHB production. However, further genetic engineering approaches are needed to make non-native microbial hosts more suitable for large-scale PHB production.

Dna2bit: high performance genomic distance estimation software for microbial genome analysis

dna2bit is an ultra-fast software specifically engineered for microbial genome analysis, particularly adept at calculating genome distances within metagenome and single amplified genome datasets. Distinguished from existing software such as Mash and Dashing, dna2bit employs feature hashing technique and Hamming distance to achieve enhanced speed and memory utilization, without sacrifice in the accuracy of average nucleotide identity calculations. dna2bit has promising applications in various domains such as average nucleotide identity approximation, metagenomic sequence clustering, and homology querying. dna2bit significantly boosts computational efficiency in handling large datasets including single amplified genomes, thereby facilitating a better understanding of the population heterogeneity and comparative genomics of microorganisms. dna2bit is available at https://github.com/lijuzeng/dna2bit.

Adaptive Laboratory Evolution of Flavin Functionality Identifies Dihydrolipoyl Dehydrogenase as One of the Critical Points for the Activity of 7,8-Didemethyl-Riboflavin as a Surrogate for Riboflavin in Escherichia coli.

Riboflavin analogs lacking one methyl group (7α or 8α) can still serve as a surrogate for riboflavin in riboflavin-deficient microorganisms or animals. The absence of both methyl groups at once completely abolishes this substitution capability. To elucidate the molecular mechanisms behind this phenomenon, we performed an adaptive laboratory evolution experiment (in triplicate) on an E. coli strain auxotrophic for riboflavin. As a result, the riboflavin requirement of the E. coli strain was reduced ~10-fold in the presence of 7,8-didemethyl-riboflavin. The whole genome sequencing of E. coli strains isolated from three experiments revealed two mutation hotspots: lpdA coding for the flavoenzyme dihydrolipoyl dehydrogenase (LpdA), and ompF coding for the major outer membrane protein. In order to investigate the essentiality of flavin's methyl groups to LpdA, the wild type and mutant variants of lpdA were cloned. At least two lpdA mutants increased the fitness of E. coli, and when 7,8-didemethyl-flavin was added to the growth medium, the increase was significant. To the best of our knowledge, an adaptive laboratory evolution experiment running in triplicate as a tool for the identification of mutation hotspots in the genome of microorganisms exposed to metabolic stress challenges is described here for the first time.

Relationship between the gut microbiome and endometriosis and its role in pathogenesis, diagnosis, and treatment: a systematic review.

Endometriosis is a chronic inflammatory disease affecting approximately 10% of women. It is defined as endometrial tissue outside of the uterus and produces a variety of symptoms including pelvic pain, dysmenorrhea, dyspareunia, and intermenstrual bleeding. Although several theories have been postulated regarding the pathogenesis of endometriosis, no theory has provided a complete explanation, therefore limiting our progress in diagnostic tools and management of endometriosis. Recently, much attention has been paid to the importance and role of the gut microbiome in endometriosis. As defined by Joshua Lederberg - microbiome is a set of the genome of microorganisms inhabiting a human body, including commensal, symbiotic and pathogenic microorganisms. The aim of this systematic review was to conduct a search in the Embase, Medline, and PubMed databases for literature from July 2013 to July 2023 regarding the relationship between the gut microbiome and endometriosis. 147 records were screened, of which 26 met the eligibility criteria, and 16 were included in this review. Our review concludes that patients with endometriosis show an altered gut microbiome, and that this has the potential to provide insight for pathogenesis, markers for diagnosis, as well as therapeutic options for treatment of endometriosis. Future research is necessary to confirm this and further investigate the relationship between the gut microbiome and endometriosis.

Metagenome from ACT-3/CF: an anaerobic chloroform-degrading microbial community.

Here, we present the metagenome of an anaerobic chloroform respiring mixed microbial community used for bioremediation. Our objective was to obtain draft genomes of key microorganisms in the culture.

Solving genomic puzzles: computational methods for metagenomic binning.

Metagenomics involves the study of genetic material obtained directly from communities of microorganisms living in natural environments. The field of metagenomics has provided valuable insights into the structure, diversity and ecology of microbial communities. Once an environmental sample is sequenced and processed, metagenomic binning clusters the sequences into bins representing different taxonomic groups such as species, genera, or higher levels. Several computational tools have been developed to automate the process of metagenomic binning. These tools have enabled the recovery of novel draft genomes of microorganisms allowing us to study their behaviors and functions within microbial communities. This review classifies and analyzes different approaches of metagenomic binning and different refinement, visualization, and evaluation techniques used by these methods. Furthermore, the review highlights the current challenges and areas of improvement present within the field of research.

Metagenomic sequences from anaerobic chloroform and dichloromethane degrading microbial communities.

Here, we present metagenomes from two cultures derived from an anaerobic microbial consortium used for bioremediation. One culture dechlorinates chloroform to dichloromethane, which is further mineralized to CO2. A second subculture was amended with only dichloromethane. We sought draft genomes of key microorganisms to identify metabolic potential in these consortia.

Functional Characterization of Endo- and Exo-Hydrolase Genes in Arabinan Degradation Gene Cluster of Bifidobacterium longum subsp. suis.

Bifidobacteria are probiotic microorganisms commonly found in the gastrointestinal tract, some of which are known to utilize linear arabino-oligosaccharides (AOS) as prebiotic carbohydrates. In general, the synergistic actions of exo-type α-l-arabinofuranosidases (ABFs) and endo-α-1,5-l-arabinanases (ABNs) are required for efficient arabinan degradation. In this study, the putative gene cluster for arabinan degradation was discovered in the genome of Bifidobacterium longum subsp. suis. It consists of a variety of genes encoding exo- and endo-hydrolases, sugar-binding proteins, ABC-binding cassettes, and transcriptional regulators. Among them, two endo-ABNs GH43 (BflsABN43A and BflsABN43B), two exo-ABFs GH43 (BflsABF43A and BflsABF43B), and an exo-ABF GH51 (BflsABF51) were predicted to be the key hydrolases for arabinan degradation. These hydrolase genes were functionally expressed in Escherichia coli, and their enzymatic properties were characterized. Their synergism in arabinan degradation has been proposed from the detailed modes of action. Extracellular endo-BflsABN43A hydrolyzes sugar beet and debranched arabinans into the short-chain branched and linear AOS. Intracellularly, AOS can be further degraded into l-arabinose via the cooperative actions of endo-BflsABN43B, exo-BflsABF43A with debranching activity, α-1,5-linkage-specific exo-BflsABF43B, and exo-BflsABF51 with dual activities. The resulting l-arabinose is expected to be metabolized into energy through the pentose phosphate pathway by three enzymes expressed from the ara operon of bifidobacteria. It is anticipated that uncovering arabinan utilization gene clusters and their detailed functions in the genomes of diverse microorganisms will facilitate the development of customized synbiotics.

ScMAR-Seq: a novel workflow for targeted single-cell genomics of microorganisms using radioactive labeling.

A central question in microbial ecology is which member of a community performs a particular metabolism. Several sophisticated isotope labeling techniques are available for analyzing the metabolic function of populations and individual cells in a community. However, these methods are generally either insufficiently sensitive or throughput-limited and thus have limited applicability for the study of complex environmental samples. Here, we present a novel approach that combines highly sensitive radioisotope tracking, microfluidics, high-throughput sorting, and single-cell genomics to simultaneously detect and identify individual microbial cells based solely on their in situ metabolic activity, without prior information on community structure.

The next-generation tools for risk assessment and precision food safety in the One Health continuum

Abstract Sequencing technologies and bioinformatics are transforming microbiology and their applications, including aspects of food quality and precision food safety and description of microbial communities in the One Health context. The use of these protocols includes, among others, a deep understanding of the genomes of microorganisms in pure culture using whole genome sequencing and importantly, metagenomics has allowed the extensive comprehension of the microbiota and microbiome of samples. Microbial communities from different sources (clinical samples, environment, food-producing animals) have increasingly been studied to describe the genetic diversity, functionality, and succession of food-borne pathogens and for studying functional microorganisms used for producing novel foods and also using fermentation. This study presents the methods used for the description of microbial communities in a model fermented food, including culturing with a novel in situ culturing approach, full-length 16S rRNA gene sequencing, and shotgun metagenomic sequencing with computational enrichment. Several microorganisms were identified both in culture and as high-quality metagenome-assembled genomes and included prominent probiotic species of the genus Gluconobacter, Liquorilactobacillus, Lactiplantibacillus, Lentilactobacillus and Lacticaseibacillus that have potential health benefits and applications in food production and environment health enhancement. These tools can revolutionise risk assessment approaches, allowing for the pinpointing of contamination events, pathogens, antimicrobial resistance spread, and description of unknown beneficial microorganisms, as in this model study. The integration of these methods in the One Health continuum will allow the precise connection of samples from clinical settings, animals, plants, and their shared environment, achieving a holistic approach to food safety and public health. Key messages • technologies are changing food safety by enabling comprehensive analysis of microbiome. This can aid in identifying contamination events, pathogens, and AMR spread, and promote One Health integration. • Use of sequencing in foods, enables the identification of novel microbial species, providing valuable insights into the functional microorganisms for production and their potential health benefits.

Improving Protection to Prevent Bacterial Infections: Preliminary Applications of Reverse Vaccinology against the Main Cystic Fibrosis Pathogens.

Reverse vaccinology is a powerful tool that was recently used to develop vaccines starting from a pathogen genome. Some bacterial infections have the necessity to be prevented then treated. For example, individuals with chronic pulmonary diseases, such as Cystic Fibrosis, are prone to develop infections and biofilms in the thick mucus that covers their lungs, mainly caused by Burkholderia cepacia complex, Haemophilus influenzae, Mycobacterium abscessus complex, Pseudomonas aeruginosa and Staphylococcus aureus. These infections are complicated to treat and prevention remains the best strategy. Despite the availability of vaccines against some strains of those pathogens, it is necessary to improve the immunization of people with Cystic Fibrosis against all of them. An effective approach is to develop a broad-spectrum vaccine to utilize proteins that are well conserved across different species. In this context, reverse vaccinology, a method based on computational analysis of the genome of various microorganisms, appears as one of the most promising tools for the identification of putative targets for broad-spectrum vaccine development. This review provides an overview of the vaccines that are under development by reverse vaccinology against the aforementioned pathogens, as well as the progress made so far.

Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics

Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment.

Exploring the Potential of Metatranscriptomics to Describe Microbial Communities and Their Effects in Molluscs.

Metatranscriptomics has emerged as a very useful technology for the study of microbiomes from RNA-seq reads. This method provides additional information compared to the sequencing of ribosomal genes because the gene expression can also be analysed. In this work, we used the metatranscriptomic approach to study the whole microbiome of mussels, including bacteria, viruses, fungi, and protozoans, by mapping the RNA-seq reads to custom assembly databases (including the genomes of microorganisms publicly available). This strategy allowed us not only to describe the diversity of microorganisms but also to relate the host transcriptome and microbiome, finding the genes more affected by the pathogen load. Although some bacteria abundant in the metatranscriptomic analysis were undetectable by 16S rRNA sequencing, a common core of the taxa was detected by both methodologies (62% of the metatranscriptomic detections were also identified by 16S rRNA sequencing, the Oceanospirillales, Flavobacteriales and Vibrionales orders being the most relevant). However, the differences in the microbiome composition were observed among different tissues of Mytilus galloprovincialis, with the fungal kingdom being especially diverse, or among molluscan species. These results confirm the potential of a meta-analysis of transcriptome data to obtain new information on the molluscs' microbiome.

Genital Microbiota and Outcome of Assisted Reproductive Treatment-A Systematic Review.

The balance between different bacterial species is essential for optimal vaginal health. Microbiome includes the host genome along with microorganism genomes and incorporates the biotic and abiotic factors, reflecting the habitat as a whole. A significant difference exists in the composition and number of the human microbiota in healthy individuals. About one-tenth of the total body microbiota exists in the urogenital tract and these can be identified by microscopy and culture-based methods, quantitative PCR, next generation and whole genome sequencing. The trend of delaying the planning of pregnancy to a later age nowadays has resulted in magnifying the use of assisted reproductive treatment (ART). Hence, genital microbiota and its impact on fertility has generated immense interest in recent years. In this systematic review, we searched the available evidence on the microbiota of the genital tract in women undergoing ART and studied the outcomes of IVF in different microbial compositions. Despite the inconsistency of the studies, it is evident that vaginal, cervical and endometrial microbiota might play a role in predicting ART outcomes. However, there is no clear evidence yet on whether the diversity, richness, quantity, or composition of species in the maternal genital tract significantly affects the outcomes in ARTs.

GenomeRxiv: a microbial whole-genome database and diagnostic marker design resource for classification, identification, and data sharing

genomeRxiv is a newly-funded US-UK collaboration to provide a public, web-accessible database of public genome sequences, accurately catalogued and classified by whole-genome similarity independent of their taxonomic affiliation. Our goal is to supply the basic and applied research community with rapid, precise and accurate identification of unknown isolates based on genome sequence alone, and with molecular tools for environmental analysis. The DNA sequencing revolution enabled the use of cultured and uncultured microorganism genomes for fast and precise identification. However, precise identification is impossible without 1. reference databases that precisely circumscribe classes of microorganisms, and label these with their uniquely-shared characteristics 2. fast algorithms that can handle the volumes of genome data Our approach integrates the highly-resolved classification framework of Life Identification Numbers (LINs) with the speed and computational efficiency of sourmash and k-mer hashing algorithms, and the precision and filtering of average nucleotide identity (ANI). We aim to construct a single genome-based indexing scheme that extends from phylum to strain, enabling the unique and consistent placement of any sequenced prokaryote genome. genomeRxiv includes protocols for confidentiality, allowing groups to identify and announce the identities of newly-sequenced organisms without sharing genome data directly. This protects communities working with commercially- and ethically-sensitive organisms (e.g. production engineering strains, potential bioweapons, and to enable benefit sharing with indigenous communities). genomeRxiv will also provide online capability to design molecular diagnostic tools for metabarcoding and qPCR, to enable tracking of specific groupings of bacteria directly in the environment.

Generation and application of pseudo-long reads for metagenome assembly.

BackgroundMetagenomic assembly using high-throughput sequencing data is a powerful method to construct microbial genomes in environmental samples without cultivation. However, metagenomic assembly, especially when only short reads are available, is a complex and challenging task because mixed genomes of multiple microorganisms constitute the metagenome. Although long read sequencing technologies have been developed and have begun to be used for metagenomic assembly, many metagenomic studies have been performed based on short reads because the generation of long reads requires higher sequencing cost than short reads.ResultsIn this study, we present a new method called PLR-GEN. It creates pseudo–long reads from metagenomic short reads based on given reference genome sequences by considering small sequence variations existing in individual genomes of the same or different species. When applied to a mock community data set in the Human Microbiome Project, PLR-GEN dramatically extended short reads in length of 101 bp to pseudo–long reads with N50 of 33 Kbp and 0.4% error rate. The use of these pseudo–long reads generated by PLR-GEN resulted in an obvious improvement of metagenomic assembly in terms of the number of sequences, assembly contiguity, and prediction of species and genes.ConclusionsPLR-GEN can be used to generate artificial long read sequences without spending extra sequencing cost, thus aiding various studies using metagenomes.

Whole genome sequencing of cyanobacterium Nostoc sp. CCCryo 231-06 using microfluidic single cell technology

SummaryThe Nostoc sp. strain CCCryo 231-06 is a cyanobacterial strain capable of surviving under extreme conditions and thus is of great interest for the astrobiology community. The knowledge of its complete genome sequence would serve as a guide for further studies. However, a major concern has been placed on the effects of contamination on the quality of sequencing data without a reference genome. Here, we report the use of microfluidic technology combined with single cell sequencing and de novo assembly to minimize the contamination and recover the complete genome of the Nostoc strain CCCryo 231-06 with high quality. 100% of the whole genome was recovered with all contaminants removed and a strongly supported phylogenetic tree. The data reported can be useful for comparative genomics for phylogenetic and taxonomic studies. The method used in this work can be applied to studies that require high-quality assemblies of genomes of unknown microorganisms.

Whole-genome sequence analysis through online web interfaces: a review.

The recent development of whole-genome sequencing technologies paved the way for understanding the genomes of microorganisms. Every whole-genome sequencing (WGS) project requires a considerable cost and a massive effort to address the questions at hand. The final step of WGS is data analysis. The analysis of whole-genome sequence is dependent on highly sophisticated bioinformatics tools that the research personal have to buy. However, many laboratories and research institutions do not have the bioinformatics capabilities to analyze the genomic data and therefore, are unable to take maximum advantage of whole-genome sequencing. In this aspect, this study provides a guide for research personals on a set of bioinformatics tools available online that can be used to analyze whole-genome sequence data of bacterial genomes. The web interfaces described here have many advantages and, in most cases exempting the need for costly analysis tools and intensive computing resources.

Meta-omics-aided isolation of an elusive anaerobic arsenic-methylating soil bacterium

Soil microbiomes harbour unparalleled functional and phylogenetic diversity. However, extracting isolates with a targeted function from complex microbiomes is not straightforward, particularly if the associated phenotype does not lend itself to high-throughput screening. Here, we tackle the methylation of arsenic (As) in anoxic soils. As methylation was proposed to be catalysed by sulfate-reducing bacteria. However, to date, there are no available anaerobic isolates capable of As methylation, whether sulfate-reducing or otherwise. The isolation of such a microorganism has been thwarted by the fact that the anaerobic bacteria harbouring a functional arsenite S-adenosylmethionine methyltransferase (ArsM) tested to date did not methylate As in pure culture. Additionally, fortuitous As methylation can result from the release of non-specific methyltransferases upon lysis. Thus, we combined metagenomics, metatranscriptomics, and metaproteomics to identify the microorganisms actively methylating As in anoxic soil-derived microbial cultures. Based on the metagenome-assembled genomes of microorganisms expressing ArsM, we isolated Paraclostridium sp. strain EML, which was confirmed to actively methylate As anaerobically. This work is an example of the application of meta-omics to the isolation of elusive microorganisms.

Conditional gene silencing in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Since the release in 2005 of the genome sequence and annotation of the first Antarctic marine bacterium, the number of genomes of psychrophilic microorganisms in public databases has steadily increased. Unfortunately, the lack of effective molecular tools for the manipulation of these environmental strains still hampers our understanding of their peculiar strategies to thrive in freezing conditions, limiting the functional genomics approaches to differential analyses only. Over the past two decades, our research group established the first effective gene cloning/expression technology in the Antarctic Gram-negative marine bacterium Pseudoalteromonas haloplanktis TAC125. The setup of a genome mutagenesis technique (based on homologous recombination and counterselection events) further supported the use of this strain, which became an attractive model for studying microbial adaptations to freezing lifestyle. Moreover, to further extend the functional analyses to its essential genes, the set-up of a conditional gene silencing approach is desirable. In this paper, we report the development of an asRNA regulatory system in the Antarctic bacterium, testing the feasibility of Hfq-dependent and PTasRNA strategies previously developed in Escherichia coli. Stable and efficient silencing of two chromosomal genes was obtained by using PTasRNAs, reaching very high levels of downregulation.