Bacterial Genome Sequences Research Articles

The hidden enzymology of bacterial natural product biosynthesis.

Bacterial natural products display astounding structural diversity, which, in turn, endows them with a remarkable range of biological activities that are of significant value to modern society. Such structural features are generated by biosynthetic enzymes that construct core scaffolds or perform peripheral modifications, and can thus define natural product families, introduce pharmacophores and permit metabolic diversification. Modern genomics approaches have greatly enhanced our ability to access and characterize natural product pathways via sequence-similarity-based bioinformatics discovery strategies. However, many biosynthetic enzymes catalyse exceptional, unprecedented transformations that continue to defy functional prediction and remain hidden from us in bacterial (meta)genomic sequence data. In this Review, we highlight exciting examples of unusual enzymology that have been uncovered recently in the context of natural product biosynthesis. These suggest that much of the natural product diversity, including entire substance classes, awaits discovery. New approaches to lift the veil on the cryptic chemistries of the natural product universe are also discussed.

In-depth comparison of library pooling strategies for multiplexing bacterial species in NGS

For bacterial genome sequencing, libraries from different strains are usually multiplexed in a single run. Normalized libraries are most often pooled in equal volumes, as recommended by next-generation sequencing platform manufacturers. This equal-volume strategy is well suited for multiplexing isolates from the same species. However, for runs involving multiple microbial species, an equimolar library pooling is more adapted because of the variation in bacterial genome size. To demonstrate its utility in clinical microbiology, we compared both equal-volume and equimolar strategies using a menu comprising 13 bacterial species involved in healthcare-associated infections. We show that equimolar pooling limits the retesting risk due to insufficient coverage depth, particularly when interspecies genome size difference is more than 2-fold. The use of this alternative strategy for multiplexing pathogenic bacteria should lead to more cost effective whole-genome sequencing applications in clinical microbiology.

RSeqTU-A Machine-Learning Based R Package for Prediction of Bacterial Transcription Units.

A transcription unit (TU) is composed of one or multiple adjacent genes on the same strand that are co-transcribed in mostly prokaryotes. Accurate identification of TUs is a crucial first step to delineate the transcriptional regulatory networks and elucidate the dynamic regulatory mechanisms encoded in various prokaryotic genomes. Many genomic features, for example, gene intergenic distance, and transcriptomic features including continuous and stable RNA-seq reads count signals, have been collected from a large amount of experimental data and integrated into classification techniques to computationally predict genome-wide TUs. Although some tools and web servers are able to predict TUs based on bacterial RNA-seq data and genome sequences, there is a need to have an improved machine learning prediction approach and a better comprehensive pipeline handling QC, TU prediction, and TU visualization. To enable users to efficiently perform TU identification on their local computers or high-performance clusters and provide a more accurate prediction, we develop an R package, named rSeqTU. rSeqTU uses a random forest algorithm to select essential features describing TUs and then uses support vector machine (SVM) to build TU prediction models. rSeqTU (available at https://s18692001.github.io/rSeqTU/) has six computational functionalities including read quality control, read mapping, training set generation, random forest-based feature selection, TU prediction, and TU visualization.

The Presence and Localization of G-Quadruplex Forming Sequences in the Domain of Bacteria.

The role of local DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, the significance of G-quadruplexes was demonstrated in the last decade, and their presence and functional relevance has been demonstrated in many genomes, including humans. In this study, we analyzed the presence and locations of G-quadruplex-forming sequences by G4Hunter in all complete bacterial genomes available in the NCBI database. G-quadruplex-forming sequences were identified in all species, however the frequency differed significantly across evolutionary groups. The highest frequency of G-quadruplex forming sequences was detected in the subgroup Deinococcus-Thermus, and the lowest frequency in Thermotogae. G-quadruplex forming sequences are non-randomly distributed and are favored in various evolutionary groups. G-quadruplex-forming sequences are enriched in ncRNA segments followed by mRNAs. Analyses of surrounding sequences showed G-quadruplex-forming sequences around tRNA and regulatory sequences. These data point to the unique and non-random localization of G-quadruplex-forming sequences in bacterial genomes.

AntiSMASH 5.0: updates to the secondary metabolite genome mining pipeline.

Secondary metabolites produced by bacteria and fungi are an important source of antimicrobials and other bioactive compounds. In recent years, genome mining has seen broad applications in identifying and characterizing new compounds as well as in metabolic engineering. Since 2011, the ‘antibiotics and secondary metabolite analysis shell—antiSMASH’ (https://antismash.secondarymetabolites.org) has assisted researchers in this, both as a web server and a standalone tool. It has established itself as the most widely used tool for identifying and analysing biosynthetic gene clusters (BGCs) in bacterial and fungal genome sequences. Here, we present an entirely redesigned and extended version 5 of antiSMASH. antiSMASH 5 adds detection rules for clusters encoding the biosynthesis of acyl-amino acids, β-lactones, fungal RiPPs, RaS-RiPPs, polybrominated diphenyl ethers, C-nucleosides, PPY-like ketones and lipolanthines. For type II polyketide synthase-encoding gene clusters, antiSMASH 5 now offers more detailed predictions. The HTML output visualization has been redesigned to improve the navigation and visual representation of annotations. We have again improved the runtime of analysis steps, making it possible to deliver comprehensive annotations for bacterial genomes within a few minutes. A new output file in the standard JavaScript object notation (JSON) format is aimed at downstream tools that process antiSMASH results programmatically.

A new statistic for efficient detection of repetitive sequences.

Detecting sequences containing repetitive regions is a basic bioinformatics task with many applications. Several methods have been developed for various types of repeat detection tasks. An efficient generic method for detecting most types of repetitive sequences is still desirable. Inspired by the excellent properties and successful applications of the D2 family of statistics in comparative analyses of genomic sequences, we developed a new statistic D2R that can efficiently discriminate sequences with or without repetitive regions. Using the statistic, we developed an algorithm of linear time and space complexity for detecting most types of repetitive sequences in multiple scenarios, including finding candidate clustered regularly interspaced short palindromic repeats regions from bacterial genomic or metagenomics sequences. Simulation and real data experiments show that the method works well on both assembled sequences and unassembled short reads. The codes are available at https://github.com/XuegongLab/D2R_codes under GPL 3.0 license. Supplementary data are available at Bioinformatics online.

English

A new method of screening type IIG restriction modification (RM) enzyme has been developed using REBASE, a database of all known and putative restriction enzymes and methyltransferases found throughout the bacterial genome sequences available in GENBANK. The in silico analysis of a group of putative type IIG RM enzymes in Microcystis aeruginosa showed a high sequence homology at both ends. This peculiarity allows for primers designing that can be used in polymerase chain reaction (PCR) to amplify the corresponding genes out of one environmental DNA extracted from a cyanobacteria-rich sample. PCR products were cloned into the pSAPV6 vector. Among eight recombinant DNA sequenced, five showed different sequences in the protein regions that interact specifically with DNA.. These five recombinant proteins expressed type IIG RM enzyme activity. Their specificities were determined, and all correspond to new DNA recognition sites. Key words: Environmental DNA, polymerase chain reaction (PCR), recombinant protein, Type IIG RM enzyme screening, uncultured bacteria.

Identification of bacterial isolates from a public hospital in Australia using complexity-reduced genotyping

Bacterial identification methods used in routine identification of pathogens in medical microbiology include a combination approach of biochemical tests, mass spectrometry or molecular biology techniques. Extensive publicly-available databases of DNA sequence data from pathogenic bacteria have been amassed in recent years; this provides an opportunity for using bacterial genome sequencing for identification purposes. Whole genome sequencing is increasing in popularity, although at present it remains a relatively expensive approach to bacterial identification and typing. Complexity-reduced bacterial genome sequencing provides an alternative. We evaluate genomic complexity-reduction using restriction enzymes and sequencing to identify bacterial isolates. A total of 165 bacterial isolates from hospital patients in the Australian Capital Territory, between 2013 and 2015 were used in this study. They were identified and typed by the Microbiology Department of Canberra Public Hospital, and represented 14 bacterial species. DNA extractions from these samples were processed using a combination of the restriction enzymes PstI with MseI, PstI with HpaII and MseI with HpaII. The resulting sequences (length 30–69 bp) were aligned against publicly available bacterial genome and plasmid sequences. Results of the alignment were processed using a bioinformatics pipeline developed for this project, Currito3.1 DNA Fragment Analysis Software. All 165 samples were correctly identified to genus and species by each of the three combinations of restriction enzymes. A further 35 samples typed to the level of strain identified and compared for consistency with MLST typing data and in silico MLST data derived from the nearest sequenced candidate reference. The high level of agreement between bacterial identification using complexity-reduced genome sequencing and standard hospital identifications indicating that this new approach is a viable alternative for identification of bacterial isolates derived from pathology specimens. The effectiveness of species identification and in particular, strain typing, depends on access to a comprehensive and taxonomically accurate bacterial genome sequence database containing relevant bacterial species and strains.

Creating cell and animal models of human disease by genome editing using CRISPR/Cas9.

A set of unique sequences in bacterial genomes, responsible for protecting bacteria against bacteriophages, has recently been used for the genetic manipulation of specific points in the genome. These systems consist of one RNA component and one enzyme component, known as CRISPR ("clustered regularly interspaced short palindromic repeats") and Cas9, respectively. The present review focuses on the applications of CRISPR/Cas9 technology in the development of cellular and animal models of human disease. Making a desired genetic alteration depends on the design of RNA molecules that guide endonucleases to a favorable genomic location. With the discovery of CRISPR/Cas9 technology, researchers are able to achieve higher levels of accuracy because of its advantages over alternative methods for editing genome, including a simple design, a high targeting efficiency and the ability to create simultaneous alterations in multiple sequences. These factors allow the researchers to apply this technology to creating cellular and animal models of human diseases by knock-in, knock-out and Indel mutation strategies, such as for Huntington's disease, cardiovascular disorders and cancers. Optimized CRISPR/Cas9 technology will facilitate access to valuable novel cellular and animal genetic models with respect to the development of innovative drug discovery and gene therapy.

A step-by-step beginner's protocol for whole genome sequencing of human bacterial pathogens.

Bacterial whole genome sequencing (WGS) is becoming a widely-used technique in research, clinical diagnostic, and public health laboratories. It enables high resolution characterization of bacterial pathogens in terms of properties that include antibiotic resistance, molecular epidemiology, and virulence. The introduction of next-generation sequencing instrumentation has made WGS attainable in terms of costs. However, the lack of a beginner’s protocol for WGS still represents a barrier to its adoption in some settings. Here, we present detailed step-by-step methods for obtaining WGS data from a range of different bacteria (Gram-positive, Gram-negative, and acid-fast) using the Illumina platform. Modifications have been performed with respect to DNA extraction and library normalization to maximize the output from the laboratory consumables invested. The protocol represents a simplified and reproducible method for producing high quality sequencing data. The key advantages of this protocol include: simplicity of the protocol for users with no prior genome sequencing experience and reproducibility of the protocol across a wide range of bacteria.

Evolutionary design principles in metabolism.

Microorganisms are ubiquitous and adapt to various dynamic environments to sustain growth. These adaptations accumulate, generating new traits forming the basis of evolution. Organisms adapt at various levels, such as gene regulation, signalling, protein-protein interactions and metabolism. Of these, metabolism forms the integral core of an organism for maintaining the growth and function of a cell. Therefore, studying adaptations in metabolic networks is crucial to understand the emergence of novel metabolic capabilities. Metabolic networks, composed of enzyme-catalysed reactions, exhibit certain repeating paradigms or design principles that arise out of different selection pressures. In this review, we discuss the design principles that are known to exist in metabolic networks, such as functional redundancy, modularity, flux coupling and exaptations. We elaborate on the studies that have helped gain insights highlighting the interplay of these design principles and adaptation. Further, we discuss how evolution plays a role in exploiting such paradigms to enhance the robustness of organisms. Looking forward, we predict that with the availability of ever-increasing numbers of bacterial, archaeal and eukaryotic genomic sequences, novel design principles will be identified, expanding our understanding of these paradigms shaped by varied evolutionary processes.

Comparative pangenomics of Campylobacter species

Bacterial genome sequencing has become very popular over the last 15 years in the field of bacterial genomics and evolution with over >1000 genome sequences generated for the zoonotic Campylobacter jejuni and Campylobacter coli species. The rapid improvement of culture and sequencing techniques has led to isolation and sequencing of new Campylobacter species from a variety of sources including birds, mammals, reptiles and the environment. Inter-species recombination has been demonstrated for C. jejuni and C. coli, but little is known about recombination between other species in the genus. We analysed the population genomic structure of whole genome sequenced isolates from different Campylobacter species using pangenome and phylogenetic analyses to investigate core and accessory gene variation and putative gene function. Characterizing the extend of genome segregation among multiple Campylobacter species isolated from the same and different hosts improved understanding of how ecology (physical isolation) maintains species and the extent to which intrinsic mechanistic and adaptive barriers are eroded when species cohabit. This broadly defines the limit of interspecies recombination and the non-reducible pangenomically-defined species. This raises important questions about the nature of species specific recombinational barriers, the genes that constitute the inter-species mobilome and the emergence of zoonoses through reticulate evolution in agricultural animals.

Discovery, properties, and biosynthesis of pseudouridimycin, an antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase.

Pseudouridimycin (PUM) is a novel pseudouridine-containing peptidyl-nucleoside antibiotic that inhibits bacterial RNA polymerase (RNAP) through a binding site and mechanism different from those of clinically approved RNAP inhibitors of the rifamycin and lipiarmycin (fidaxomicin) classes. PUM was discovered by screening microbial fermentation extracts for RNAP inhibitors. In this review, we describe the discovery and characterization of PUM. We also describe the RNAP-inhibitory and antibacterial properties of PUM. Finally, we review available information on the gene cluster and pathway for PUM biosynthesis and on the potential for discovering additional novel pseudouridine-containing nucleoside antibiotics by searching bacterial genome and metagenome sequences for sequences similar to pumJ, the pseudouridine-synthase gene of the PUM biosynthesis gene cluster.

Complete genome sequence of the halophilic PHA-producing bacterium Halomonas sp. SF2003: insights into its biotechnological potential.

A halophilic Gram-negative eubacterium was isolated from the Iroise Sea and identified as an efficient producer of polyhydroxyalkanoates (PHA). The strain, designated SF2003, was found to belong to the Halomonas genus on the basis of 16S rRNA gene sequence similarity. Previous biochemical tests indicated that the Halomonas sp. strain SF2003 is capable of supporting various culture conditions which sometimes can be constraining for marine strains. This versatility could be of great interest for biotechnological applications. Therefore, a complete bacterial genome sequencing and de novo assembly were performed using a PacBio RSII sequencer and Hierarchical Genome Assembly Process software in order to predict Halomonas sp. SF2003 metabolisms, and to identify genes involved in PHA production and stress tolerance. This study demonstrates the complete genome sequence of Halomonas sp. SF2003 which contains a circular 4,36 Mbp chromosome, and replaces the strain in a phylogenetic tree. Genes related to PHA metabolism, carbohydrate metabolism, fatty acid metabolism and stress tolerance were identified and a comparison was made with metabolisms of relative species. Genes annotation highlighted the presence of typical genes involved in PHA biosynthesis such as phaA, phaB and phaC and enabled a preliminary analysis of their organization and characteristics. Several genes of carbohydrates and fatty acid metabolisms were also identified which provided helpful insights into both a better knowledge of the intricacies of PHA biosynthetic pathways and of production purposes. Results show the strong versatility of Halomonas sp. SF2003 to adapt to various temperatures and salinity which can subsequently be exploited for industrial applications such as PHA production.

Highly conserved nucleotide motifs present in the 5'UTR of the heme-receptor gene shmR are required for HmuP-dependent expression of shmR in Ensifer meliloti.

Heme may represent a major iron-source for bacteria. In the symbiotic nitrogen-fixing bacterium Ensifer meliloti 1021, iron acquisition from heme depends on the outer-membrane heme-receptor ShmR. Expression of shmR gene is repressed by iron in a RirA dependent manner while under iron-limitation its expression requires the small protein HmuP. In this work, we identified highly conserved nucleotide motifs present upstream the shmR gene. These motifs are widely distributed among Alpha and Beta Proteobacteria, and correlate with the presence of HmuP coding sequences in bacterial genomes. According to data presented in this work, we named these new motifs as HmuP-responsive elements (HPREs). In the analyzed genomes, the HPREs were always present upstream of genes encoding putative heme-receptors. Moreover, in those Alpha and Beta Proteobacteria where transcriptional start sites for shmR homologs are known, HPREs were located in the 5'UTR region. In this work we show that in E. meliloti 1021, HPREs are involved in HmuP-dependent shmR expression. Moreover, we show that changes in sequence composition of the HPREs correlate with changes in a predicted RNA secondary structure element and affect shmR gene expression.

Ultrafast search of all deposited bacterial and viral genomic data.

Exponentially increasing amounts of unprocessed bacterial and viral genomic sequence data are stored in the global archives. The ability to query these data for sequence search-terms would facilitate both basic research and applications such as real-time genomic epidemiology and surveillance. However, this is not possible with current methods. To solve this problem, we combine knowledge of microbial population genomics with computational methods devised for web-search to produce a searchable data structure named Bitsliced Genomic Signature Index (BIGSI). We indexed the entire global corpus of 447,833 bacterial and viral whole genome sequence datasets using 4 orders of magnitude less storage than previous methods. We applied our BIGSI search function to rapidly find resistance genes MCR-1/2/3, determine the host-range of 2827 plasmids, and quantify antibiotic resistance in archived datasets. Our index can grow incrementally as new (unprocessed or assembled) sequence datasets are deposited and can scale to millions of datasets.

A human gut bacterial genome and culture collection for improved metagenomic analyses

Understanding gut microbiome functions requires cultivated bacteria for experimental validation and reference bacterial genome sequences to interpret metagenome datasets and guide functional analyses. We present the Human Gastrointestinal Bacteria Culture Collection (HBC), a comprehensive set of 737 whole-genome-sequenced bacterial isolates, representing 273 species (105 novel species) from 31 families found in the human gastrointestinal microbiota. The HBC increases the number of bacterial genomes derived from human gastrointestinal microbiota by 37%. The resulting global Human Gastrointestinal Bacteria Genome Collection (HGG) classifies 83% of genera by abundance across 13,490 shotgun-sequenced metagenomic samples, improves taxonomic classification by 61% compared to the Human Microbiome Project (HMP) genome collection and achieves subspecies-level classification for almost 50% of sequences. The improved resource of gastrointestinal bacterial reference sequences circumvents dependence on de novo assembly of metagenomes and enables accurate and cost-effective shotgun metagenomic analyses of human gastrointestinal microbiota.

Compositional assessment of bacterial communities in probiotic supplements by means of metagenomic techniques

Health promoting or probiotic bacteria are commonly incorporated into a variety of functional foods and drug formulations, due to their purported ability to confer benefit to host health. Despite the extensive commercial exploitation of probiotic formulations there are still major knowledge gaps regarding the precise molecular mechanism of action and corresponding genetic/genomic properties of probiotic bacteria. In the current study, we describe a metagenomic approach which allows determination of the composition of probiotic supplements through next-generation sequencing analyses based on rRNA-associated sequences to assess bacterial composition of the product combined with a shotgun metagenomics approach directed to decode the genome sequences of the probiotic strains for each product assayed. The here developed approach has been tested for 10 probiotic supplements, revealing inconsistencies between the identified probiotic strains and the declared strains as indicated by the producers. Furthermore, the decoded bacterial genome sequence of Bifidobacterium animalis subsp. lactis BB-12 from a 1995 frozen dried stock revealed genetic evidence for genome evolution and stability of this microorganism when compared with the re-constructed genome of the identical strain from a probiotic supplement of 2017.

Bacterial genome sequencing and analysis: paving the way for a Switzerland-wide molecular epidemiological surveillance platform.

Facing multidrug resistant (MDR) bacterial pathogens is one of the most important challenges for our society. The spread of highly virulent and resistant pathogens can be described using molecular typing technologies; in particular, whole genome sequencing (WGS) data can be usedf or molecular typing purposes with high resolution. WGS data analysis can explain the spatiotemporal patterns of pathogen transmission. However, the transmission between compartments (human, animal, food, environment) is very complex. Interoperable and curated metadata are a key requirement for fully understanding this complexity. In addition, high quality sequence data are a key element between centres using WGS data for diagnostic and epidemiological applications. We aim to describe steps to improve WGS data analysis and to implement a molecular surveillance platform allowing integration of high resolution WGS typing data and epidemiological data.

Local Symmetry of Non-Coding Genetic Sequences

The simplest hypothesis of DNA strand symmetry states that proportions of nucleotides of the same base pair are approximately equal within single DNA strands. Results of extensive empirical studies using asymmetry measures and various visualization tools show that for long DNA sequences (approximate) strand symmetry generally holds with rather rare exceptions. In the paper, a formal definition of DNA strand local symmetry is presented, characterized in terms of generalized logits and tested for the longest non-coding sequences of bacterial genomes. Validity of a special regression-type probabilistic structure of the data is supposed. This structure is compatible with probability distribution of random nucleotide sequences at a steady state of a context-dependent reversible Markov evolutionary process. The null hypothesis of strand local symmetry is rejected in majority of bacterial genomes suggesting that even neutral mutations are skewed with respect to leading and lagging strands.