While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

Pathogenic bacteria continuously encounter multiple forms of stress in their hostile environments, which leads to DNA damage. With the new insight into biology offered by genome sequences, the elucidation of the gene content encoding proteins provides clues toward understanding the microbial lifestyle related to habitat and niche. Campylobacter jejuni, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis, the pathogenic Neisseria, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus are major human pathogens causing detrimental morbidity and mortality at a global scale. An algorithm for the clustering of orthologs was established in order to identify whether orthologs of selected genes were present or absent in the genomes of the pathogenic bacteria under study. Based on the known genes for the various functions and their orthologs in selected pathogenic bacteria, an overview of the presence of the different types of genes was created. In this context, we focus on selected processes enabling genome dynamics in these particular pathogens, namely DNA repair, recombination and horizontal gene transfer. An understanding of the precise molecular functions of the enzymes participating in DNA metabolism and their importance in the maintenance of bacterial genome integrity has also, in recent years, indicated a future role for these enzymes as targets for therapeutic intervention.

Organisms have evolved ways of regulating transcription to better adapt to varying environments. Could the current functional genomics data and models support the possibility of engineering a genome with completely rearranged gene organization while the cell maintains its behavior under environmental challenges? How would we proceed to design a full nucleotide sequence for such genomes?

Among challenges that hamper reaping the benefits of genome assembly are both unfinished assemblies and the ensuing experimental costs. First, numerous software solutions for genome de novo assembly are available, each having its advantages and drawbacks, without clear guidelines as to how to choose among them. Second, these solutions produce draft assemblies that often require a resource intensive finishing phase.

Microbial interactions are expected to be major determinants of microbiome structure and function. Although fungi are found in diverse microbiomes, their interactions with bacteria remain largely uncharacterized. In this work, we characterize interactions in 16 different bacterial-fungal pairs, examining the impacts of 8 different fungi isolated from cheese rind microbiomes on 2 bacteria (Escherichia coli and a cheese-isolated Pseudomonas psychrophila). Using random barcode transposon-site sequencing with an analysis pipeline that allows statistical comparisons between different conditions, we observed that fungal partners caused widespread changes in the fitness of bacterial mutants compared to growth alone. We found that all fungal species modulated the availability of iron and biotin to bacterial species, which suggests that these may be conserved drivers of bacterial-fungal interactions. Species-specific interactions were also uncovered, a subset of which suggested fungal antibiotic production. Changes in both conserved and species-specific interactions resulted from the deletion of a global regulator of fungal specialized metabolite production. This work highlights the potential for broad impacts of fungi on bacterial species within microbiomes.

Genome structure variation has profound impacts on phenotype in organisms ranging from microbes to humans, yet little is known about how natural selection acts on genome arrangement. Pathogenic bacteria such as Yersinia pestis, which causes bubonic and pneumonic plague, often exhibit a high degree of genomic rearrangement. The recent availability of several Yersinia genomes offers an unprecedented opportunity to study the evolution of genome structure and arrangement. We introduce a set of statistical methods to study patterns of rearrangement in circular chromosomes and apply them to the Yersinia. We constructed a multiple alignment of eight Yersinia genomes using Mauve software to identify 78 conserved segments that are internally free from genome rearrangement. Based on the alignment, we applied Bayesian statistical methods to infer the phylogenetic inversion history of Yersinia. The sampling of genome arrangement reconstructions contains seven parsimonious tree topologies, each having different histories of 79 inversions. Topologies with a greater number of inversions also exist, but were sampled less frequently. The inversion phylogenies agree with results suggested by SNP patterns. We then analyzed reconstructed inversion histories to identify patterns of rearrangement. We confirm an over-representation of "symmetric inversions"-inversions with endpoints that are equally distant from the origin of chromosomal replication. Ancestral genome arrangements demonstrate moderate preference for replichore balance in Yersinia. We found that all inversions are shorter than expected under a neutral model, whereas inversions acting within a single replichore are much shorter than expected. We also found evidence for a canonical configuration of the origin and terminus of replication. Finally, breakpoint reuse analysis reveals that inversions with endpoints proximal to the origin of DNA replication are nearly three times more frequent. Our findings represent the first characterization of genome arrangement evolution in a bacterial population evolving outside laboratory conditions. Insight into the process of genomic rearrangement may further the understanding of pathogen population dynamics and selection on the architecture of circular bacterial chromosomes.

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44 other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.

Bacterial Rho-independent terminators (RITs) are important genomic landmarks involved in gene regulation and terminating gene expression. In this investigation we present RNIE, a probabilistic approach for predicting RITs. The method is based upon covariance models which have been known for many years to be the most accurate computational tools for predicting homology in structural non-coding RNAs. We show that RNIE has superior performance in model species from a spectrum of bacterial phyla. Further analysis of species where a low number of RITs were predicted revealed a highly conserved structural sequence motif enriched near the genic termini of the pathogenic Actinobacteria, Mycobacterium tuberculosis. This motif, together with classical RITs, account for up to 90% of all the significantly structured regions from the termini of M. tuberculosis genic elements. The software, predictions and alignments described below are available from http://github.com/ppgardne/RNIE.

With the exponential increase in the number of bacterial taxa with genome sequence data, a new standardized method to assign species designations is needed that is consistent with classically obtained taxonomic analyses. This is particularly acute for unculturable, obligate intracellular bacteria with which classically defined methods, like DNA-DNA hybridization, cannot be used, such as those in the Rickettsiales. In this study, we generated nucleotide-based core genome alignments for a wide range of genera with classically defined species, as well as those within the Rickettsiales. We created a workflow that uses the length, sequence identity, and phylogenetic relationships inferred from core genome alignments to assign genus and species designations that recapitulate classically obtained results. Using this method, most classically defined bacterial genera have a core genome alignment that is ≥10% of the average input genome length. Both Anaplasma and Neorickettsia fail to meet this criterion, indicating that the taxonomy of these genera should be reexamined. Consistently, genomes from organisms with the same species epithet have ≥96.8% identity of their core genome alignments. Additionally, these core genome alignments can be used to generate phylogenomic trees to identify monophyletic clades that define species and neighbor-network trees to assess recombination across different taxa. By these criteria, Wolbachia organisms are delineated into species different from the currently used supergroup designations, while Rickettsia organisms are delineated into 9 distinct species, compared to the current 27 species. By using core genome alignments to assign taxonomic designations, we aim to provide a high-resolution, robust method to guide bacterial nomenclature that is aligned with classically obtained results. IMPORTANCE With the increasing availability of genome sequences, we sought to develop and apply a robust, portable, and high-resolution method for the assignment of genera and species designations that can recapitulate classically defined taxonomic designations. Using cutoffs derived from the lengths and sequence identities of core genome alignments along with phylogenetic analyses, we sought to evaluate or reevaluate genus- and species-level designations for diverse taxa, with an emphasis on the order Rickettsiales, where species designations have been applied inconsistently. Our results indicate that the Rickettsia genus has an overabundance of species designations, that the current Anaplasma and Neorickettsia genus designations are both too broad and need to be divided, and that there are clear demarcations of Wolbachia species that do not align precisely with the existing supergroup designations.

Illumina sequencing platforms have enabled widespread bacterial whole genome sequencing. While Illumina data is appropriate for many analyses, its short read length limits its ability to resolve genomic structure. This has major implications for tracking the spread of mobile genetic elements, including those which carry antimicrobial resistance determinants. Fully resolving a bacterial genome requires long-read sequencing such as those generated by Oxford Nanopore Technologies (ONT) platforms. Here we describe our use of the ONT MinION to sequence 12 isolates of Klebsiella pneumoniae on a single flow cell. We assembled each genome using a combination of ONT reads and previously available Illumina reads, and little to no manual intervention was needed to achieve fully resolved assemblies using the Unicycler hybrid assembler. Assembling only ONT reads with Canu was less effective, resulting in fewer resolved genomes and higher error rates even following error correction with Nanopolish. We demonstrate that multiplexed ONT sequencing is a valuable tool for high-throughput bacterial genome finishing. Specifically, we advocate the use of Illumina sequencing as a first analysis step, followed by ONT reads as needed to resolve genomic structure.

The arrangement of nucleotides within a bacterial chromosome is influenced by numerous factors. The degeneracy of the third codon within each reading frame allows some flexibility of nucleotide selection; however, the third nucleotide in the triplet of each codon is at least partly determined by the preceding two. This is most evident in organisms with a strong G + C bias, as the degenerate codon must contribute disproportionately to maintaining that bias. Therefore, a correlation exists between the first two nucleotides and the third in all open reading frames. If the arrangement of nucleotides in a bacterial chromosome is represented as a Markov process, we would expect that the correlation would be completely captured by a second-order Markov model and an increase in the order of the model (e.g., third-, fourth-…order) would not capture any additional uncertainty in the process. In this manuscript, we present the results of a comprehensive study of the Markov property that exists in the DNA sequences of 906 bacterial chromosomes. All of the 906 bacterial chromosomes studied exhibit a statistically significant Markov property that extends beyond second-order, and therefore cannot be fully explained by codon usage. An unrooted tree containing all 906 bacterial chromosomes based on their transition probability matrices of third-order shares ∼25% similarity to a tree based on sequence homologies of 16S rRNA sequences. This congruence to the 16S rRNA tree is greater than for trees based on lower-order models (e.g., second-order), and higher-order models result in diminishing improvements in congruence. A nucleotide correlation most likely exists within every bacterial chromosome that extends past three nucleotides. This correlation places significant limits on the number of nucleotide sequences that can represent probable bacterial chromosomes. Transition matrix usage is largely conserved by taxa, indicating that this property is likely inherited, however some important exceptions exist that may indicate the convergent evolution of some bacteria.

The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

Increasing evidence supports the notion that different regions of a genome have unique rates of molecular change. This variation is particularly evident in bacterial genomes where previous studies have reported gene expression and essentiality tend to decrease, whereas substitution rates usually increase with increasing distance from the origin of replication. Genomic reorganization such as rearrangements occur frequently in bacteria and allow for the introduction and restructuring of genetic content, creating gradients of molecular traits along genomes. Here, we explore the interplay of these phenomena by mapping substitutions to the genomes of Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti, quantifying how many substitutions have occurred at each position in the genome. Preceding work indicates that substitution rate significantly increases with distance from the origin. Using a larger sample size and accounting for genome rearrangements through ancestral reconstruction, our analysis demonstrates that the correlation between the number of substitutions and the distance from the origin of replication is significant but small and inconsistent in direction. Some replicons had a significantly decreasing trend (E. coli and the chromosome of S. meliloti), whereas others showed the opposite significant trend (B. subtilis, Streptomyces, pSymA and pSymB in S. meliloti). dN, dS, and ω were examined across all genes and there was no significant correlation between those values and distance from the origin. This study highlights the impact that genomic rearrangements and location have on molecular trends in some bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis. Assuming that molecular trends are exclusively in one direction can be problematic.

Bacterial genomes have characteristic compositional skews, which are differences in nucleotide frequency between the leading and lagging DNA strands across a segment of a genome. It is thought that these strand asymmetries arise as a result of mutational biases and selective constraints, particularly for energy efficiency. Analysis of compositional skews in a diverse set of bacteria provides a comparative context in which mutational and selective environmental constraints can be studied. These analyses typically require finished and well-annotated genomic sequences.

Gene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods such as recombineering.

CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in E. coli at efficiencies approaching ∼100%, generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CAST systems, including guidelines on the available homologs and vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational crRNA design algorithm to avoid potential off-targets and CRISPR array cloning pipeline for DNA insertion multiplexing. Starting from available plasmid constructs, the isolation of clonal strains containing a novel genomic integration event-of-interest can be achieved in 1 week using standard molecular biology techniques.

Determining the genomic sequences of microorganisms is the basis and prerequisite for understanding their biology and functional characterization. While the advent of low-cost, extremely high-throughput second-generation sequencing technologies and the parallel development of assembly algorithms have generated rapid and cost-effective genome assemblies, such assemblies are often unfinished, fragmented draft genomes as a result of short read lengths and long repeats present in multiple copies. Third-generation, PacBio sequencing technologies circumvented this problem by greatly increasing read length. Hybrid approaches including ALLPATHS-LG, PacBio corrected reads pipeline, SPAdes, and SSPACE-LongRead, and non-hybrid approaches--hierarchical genome-assembly process (HGAP) and PacBio corrected reads pipeline via self-correction--have therefore been proposed to utilize the PacBio long reads that can span many thousands of bases to facilitate the assembly of complete microbial genomes. However, standardized procedures that aim at evaluating and comparing these approaches are currently insufficient. To address the issue, we herein provide a comprehensive comparison by collecting datasets for the comparative assessment on the above-mentioned five assemblers. In addition to offering explicit and beneficial recommendations to practitioners, this study aims to aid in the design of a paradigm positioned to complete bacterial genome assembly.

By sequencing the genomes of 34 mutation accumulation lines of a mismatch-repair defective strain of Escherichia coli that had undergone a total of 12,750 generations, we identified 1625 spontaneous base-pair substitutions spread across the E. coli genome. These mutations are not distributed at random but, instead, fall into a wave-like spatial pattern that is repeated almost exactly in mirror image in the two separately replicated halves of the bacterial chromosome. The pattern is correlated to genomic features, with mutation densities greatest in regions predicted to have high superhelicity. Superimposed upon this pattern are regional hotspots, some of which are located where replication forks may collide or be blocked. These results suggest that, as they traverse the chromosome, the two replication forks encounter parallel structural features that change the fidelity of DNA replication.

We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

The physical stability of bacterial chromosomes is important for their in vitro manipulation, while genetic stability is important in vivo. However, extracted naked chromosomes in the open circular form are fragile due to nicks and gaps. Using a nick/gap repair and negative supercoiling reaction (named SCR), we first achieved the negative supercoiling of the whole genomes extracted from Escherichia coli and Vibrio natriegens cells. Supercoiled chromosomes of 0.2-4.6 megabase (Mb) were separated by size using a conventional agarose gel electrophoresis and served as DNA size markers. We also achieved the enzymatic replication of 1-2 Mb chromosomes using the reconstituted E. coli replication-cycle reaction (RCR). Electroporation-ready 1 Mb chromosomes were prepared by a modified SCR performed at a low salt concentration (L-SCR) and directly introduced into commercial electrocompetent E. coli cells. Since successful electroporation relies on the genetic stability of a chromosome in cells, genetically stable 1 Mb chromosomes were developed according to a portable chromosome format (PCF). Using physically and genetically stabilized chromosomes, the democratization of genome synthetic biology will be greatly accelerated.