Recovery and Analysis of Long-Read Metagenome-Assembled Genomes.

The development of long-read nucleic acid sequencing is beginning to make very substantive impact on the conduct of metagenome analysis, particularly in relation to the problem of recovering the genomes of member species of complex microbial communities. Here we outline bioinformatics workflows for the recovery and characterization of complete genomes from long-read metagenome data and some complementary procedures for comparison of cognate draft genomes and gene quality obtained from short-read sequencing and long-read sequencing.

Recovery of High Quality Metagenome-Assembled Genomes From Full-Scale Activated Sludge Microbial Communities in a Tropical Climate Using Longitudinal Metagenome Sampling.

The analysis of metagenome data based on the recovery of draft genomes (so called metagenome-assembled genomes, or MAG) has assumed an increasingly central role in microbiome research in recent years. Microbial communities underpinning the operation of wastewater treatment plants are particularly challenging targets for MAG analysis due to their high ecological complexity, and remain important, albeit understudied, microbial communities that play ssa key role in mediating interactions between human and natural ecosystems. Here we consider strategies for recovery of MAG sequence from time series metagenome surveys of full-scale activated sludge microbial communities. We generate MAG catalogs from this set of data using several different strategies, including the use of multiple individual sample assemblies, two variations on multi-sample co-assembly and a recently published MAG recovery workflow using deep learning. We obtain a total of just under 9,100 draft genomes, which collapse to around 3,100 non-redundant genomic clusters. We examine the strengths and weaknesses of these approaches in relation to MAG yield and quality, showing that co-assembly may offer advantages over single-sample assembly in the case of metagenome data obtained from closely sampled longitudinal study designs. Around 1,000 MAGs were candidates for being considered high quality, based on single-copy marker gene occurrence statistics, however only 58 MAG formally meet the MIMAG criteria for being high quality draft genomes. These findings carry broader broader implications for performing genome-resolved metagenomics on highly complex communities, the design and implementation of genome recoverability strategies, MAG decontamination and the search for better binning methodology.

Comparative Genomics of Members of the Genus Defluviicoccus With Insights Into Their Ecophysiological Importance.

Members of the genus Defluviicoccus occur often at high abundances in activated sludge wastewater treatment plants designed to remove phosphorus, where biomass is subjected to alternating anaerobic feed/aerobic famine conditions, believed to favor the proliferation of organisms like Ca. Accumulibacter and other phosphate-accumulating organisms (PAO), and Defluviicoccus. All have a capacity to assimilate readily metabolizable substrates and store them intracellularly during the anaerobic feed stage so that under the subsequent famine aerobic stage, these can be used to synthesize polyphosphate reserves by the PAO and glycogen by Defluviicoccus. Consequently, Defluviicoccus is described as a glycogen-accumulating organism or GAO. Because they share a similar anaerobic phenotype, it has been proposed that at high Defluviicoccus abundance, the PAO are out-competed for assimilable metabolites anaerobically, and hence aerobic P removal capacity is reduced. Several Defluviicoccus whole genome sequences have been published (Ca. Defluviicoccus tetraformis, Defluviicoccus GAO-HK, and Ca. Defluviicoccus seviourii). The available genomic data of these suggest marked metabolic differences between them, some of which have ecophysiological implications. Here, we describe the whole genome sequence of the type strain Defluviicoccus vanusT , the only cultured member of this genus, and a detailed comparative re-examination of all extant Defluviicoccus genomes. Each, with one exception, which appears not to be a member of this genus, contains the genes expected of GAO members, in possessing multiple copies of those for glycogen biosynthesis and catabolism, and anaerobic polyhydroxyalkanoate (PHA) synthesis. Both 16S rRNA and genome sequence data suggest that the current recognition of four clades is insufficient to embrace their phylogenetic biodiversity, but do not support the view that they should be re-classified into families other than their existing location in the Rhodospirillaceae. As expected, considerable variations were seen in the presence and numbers of genes encoding properties associated with key substrate assimilation and metabolic pathways. Two genomes also carried the pit gene for synthesis of the low-affinity phosphate transport protein, pit, considered by many to distinguish all PAO from GAO. The data re-emphasize the risks associated with extrapolating the data generated from a single Defluviicoccus population to embrace all members of that genus.

Modular evolution of secretion systems and virulence plasmids in a bacterial species complex.

BACKGROUND: Many named species as defined in current bacterial taxonomy correspond to species complexes. Uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized the Agrobacterium tumefaciens species complex (a.k.a. Agrobacterium biovar 1), a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes. RESULTS: We utilized 35 genome assemblies, including 14 newly generated ones, to achieve a phylogenetically balanced sampling of A. tumefaciens. Our genomic analysis suggested that the 10 genomospecies described previously are distinct biological species and supported a quantitative guideline for species delineation. Furthermore, our inference of gene content and core-genome phylogeny allowed for investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins. CONCLUSIONS: We demonstrate that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. We show that the T6SS VgrG proteins involved in specific effector binding and delivery can be classified into distinct types based on domain organization. The co-occurrence patterns of VgrG-associated domains and the neighboring genes that encode different chaperones/effectors can be used to infer possible interacting partners. Similarly, the associations between plant host preference and the pTi type among these strains can be used to infer phenotype-genotype correspondence. Our strategies for multi-level investigations at scales that range from whole genomes to intragenic domains and phylogenetic depths from between- to within-species are applicable to other bacteria. Furthermore, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation.

BACKGROUND: Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. RESULTS: Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. CONCLUSIONS: The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.

Recovery of complete genomes and non-chromosomal replicons from activated sludge enrichment microbial communities with long read metagenome sequencing.

New long read sequencing technologies offer huge potential for effective recovery of complete, closed genomes from complex microbial communities. Using long read data (ONT MinION) obtained from an ensemble of activated sludge enrichment bioreactors we recover 22 closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses, specifically microbes known to exhibit the polyphosphate- and glycogen-accumulating organism phenotypes (namely Candidatus Accumulibacter and Dechloromonas, and Micropruina, Defluviicoccus and Candidatus Contendobacter, respectively), and filamentous bacteria (Thiothrix) associated with the formation and stability of activated sludge flocs. Additionally we demonstrate the recovery of close to 100 circularised plasmids, phages and small microbial genomes from these microbial communities using long read assembled sequence. We describe methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes, and assess the influence of different correction procedures on genome quality and predicted gene quality. Our findings establish the feasibility of performing long read metagenome-assembled genome recovery for both chromosomal and non-chromosomal replicons, and demonstrate the value of parallel sampling of moderately complex enrichment communities to obtaining high quality reference genomes of key functional species relevant for wastewater bioprocesses.

Differentiations in Gene Content and Expression Response to Virulence Induction Between Two Agrobacterium Strains.

Agrobacterium tumefaciens is important in biotechnology due to its ability to transform eukaryotic cells. Although the molecular mechanisms have been studied extensively, previous studies were focused on the model strain C58. Consequently, nearly all of the commonly used strains for biotechnology application were derived from C58 and share similar host ranges. To overcome this limitation, better understanding of the natural genetic variation could provide valuable insights. In this study, we conducted comparative analysis between C58 and 1D1609. These two strains belong to different genomospecies within the species complex and have distinct infectivity profiles. Genome comparisons revealed that each strain has >1,000 unique genes in addition to the 4,115 shared genes. Furthermore, the divergence in gene content and sequences vary among replicons. The circular chromosome is much more conserved compared to the linear chromosome. To identify the genes that may contribute to their differentiation in virulence, we compared the transcriptomes to screen for genes differentially expressed in response to the inducer acetosyringone. Based on the RNA-Seq results with three biological replicates, ∼100 differentially expressed genes were identified in each strain. Intriguingly, homologous genes with the same expression pattern account for <50% of these differentially expressed genes. This finding indicated that phenotypic variation may be partially explained by divergence in expression regulation. In summary, this study characterized the genomic and transcriptomic differences between two representative Agrobacterium strains. Moreover, the short list of differentially expressed genes are promising candidates for future characterization, which could improve our understanding of the genetic mechanisms for phenotypic divergence.

The genome of 'Candidatus Phytoplasma solani' strain SA-1 is highly dynamic and prone to adopting foreign sequences.

Bacteria of the genus 'Candidatus Phytoplasma' are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as 'Ca. P. solani', have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of 'Ca. P. solani' strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The 'Ca. P. solani' SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.

Presence of an Agrobacterium-Type Tumor-Inducing Plasmid in Neorhizobium sp. NCHU2750 and the Link to Phytopathogenicity.

The genus Agrobacterium contains a group of plant-pathogenic bacteria that have been developed into an important tool for genetic transformation of eukaryotes. To further improve this biotechnology application, a better understanding of the natural genetic variation is critical. During the process of isolation and characterization of wild-type strains, we found a novel strain (i.e., NCHU2750) that resembles Agrobacterium phenotypically but exhibits high sequence divergence in several marker genes. For more comprehensive characterization of this strain, we determined its complete genome sequence for comparative analysis and performed pathogenicity assays on plants. The results demonstrated that this strain is closely related to Neorhizobium in chromosomal organization, gene content, and molecular phylogeny. However, unlike the characterized species within Neorhizobium, which all form root nodules with legume hosts and are potentially nitrogen-fixing mutualists, NCHU2750 is a gall-forming pathogen capable of infecting plant hosts across multiple families. Intriguingly, this pathogenicity phenotype could be attributed to the presence of an Agrobacterium-type tumor-inducing plasmid in the genome of NCHU2750. These findings suggest that these different lineages within the family Rhizobiaceae are capable of transitioning between ecological niches by having novel combinations of replicons. In summary, this work expanded the genomic resources available within Rhizobiaceae and provided a strong foundation for future studies of this novel lineage. With an infectivity profile that is different from several representative Agrobacterium strains, this strain may be useful for comparative analysis to better investigate the genetic determinants of host range among these bacteria.

Complete Genome Sequence of Agrobacterium tumefaciens 1D1609.

Agrobacterium tumefaciens 1D1609 is a highly virulent strain isolated from a crown gall tumor of alfalfa (Medicago sativa L.). Compared to other well-characterized A. tumefaciens strains, such as C58 and Ach5, 1D1609 has a distinctive host range. Here, we report its complete genome sequence to facilitate future studies.

Correction: Complete chloroplast genome sequence of common bermudagrass (Cynodon dactylon (L.) Pers.) and comparative analysis within the family Poaceae.

[This corrects the article DOI: 10.1371/journal.pone.0179055.].

Complete chloroplast genome sequence of common bermudagrass (Cynodon dactylon (L.) Pers.) and comparative analysis within the family Poaceae.

Common bermudagrass (Cynodon dactylon (L.) Pers.) belongs to the subfamily Chloridoideae of the Poaceae family, one of the most important plant families ecologically and economically. This grass has a long connection with human culture but its systematics is relatively understudied. In this study, we sequenced and investigated the chloroplast genome of common bermudagrass, which is 134,297 bp in length with two single copy regions (LSC: 79,732 bp; SSC: 12,521 bp) and a pair of inverted repeat (IR) regions (21,022 bp). The annotation contains a total of 128 predicted genes, including 82 protein-coding, 38 tRNA, and 8 rRNA genes. Additionally, our in silico analyses identified 10 sets of repeats longer than 20 bp and predicted the presence of 36 RNA editing sites. Overall, the chloroplast genome of common bermudagrass resembles those from other Poaceae lineages. Compared to most angiosperms, the accD gene and the introns of both clpP and rpoC1 genes are missing. Additionally, the ycf1, ycf2, ycf15, and ycf68 genes are pseudogenized and two genome rearrangements exist. Our phylogenetic analysis based on 47 chloroplast protein-coding genes supported the placement of common bermudagrass within Chloridoideae. Our phylogenetic character mapping based on the parsimony principle further indicated that the loss of the accD gene and clpP introns, the pseudogenization of four ycf genes, and the two rearrangements occurred only once after the most recent common ancestor of the Poaceae diverged from other monocots, which could explain the unusual long branch leading to the Poaceae when phylogeny is inferred based on chloroplast sequences.

Complete Genome Sequence of Spiroplasma sp. NBRC 100390.

Spiroplasma sp. NBRC 100390 was initially described as a duplicate of S. atrichopogonis GNAT3597T (=ATCC BAA-520T) but later found to be different in the 16S rDNA sequences. Here, we report the complete genome sequence of this bacterium to establish its identity and to facilitate future investigation.

Complete Genome Sequence of Spiroplasma sp. TU-14.

Spiroplasma sp. TU-14 was isolated from a contaminated sample of Entomoplasma lucivorax PIPN-2T obtained from the International Organization for Mycoplasmology collection. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma spp.

A few sequence polymorphisms among isolates of Maize bushy stunt phytoplasma associate with organ proliferation symptoms of infected maize plants.

Background and Aims: Maize bushy stunt phytoplasma (MBSP) is a bacterial pathogen of maize ( Zea mays L.) across Latin America. MBSP belongs to the 16SrI-B sub-group within the genus ' Candidatus Phytoplasma'. MBSP and its insect vector Dalbulus maidis (Hemiptera: Cicadellidae) are restricted to maize; both are thought to have coevolved with maize during its domestication from a teosinte-like ancestor. MBSP-infected maize plants show a diversity of symptoms. and it is likely that MBSP is under strong selection for increased virulence and insect transmission on maize hybrids that are widely grown in Brazil. In this study it was investigated whether the differences in genome sequences of MBSP isolates from two maize-growing regions in South-east Brazil explain variations in symptom severity of the MBSP isolates on various maize genotypes. Methods: MBSP isolates were collected from maize production fields in Guaíra and Piracicaba in South-east Brazil for infection assays. One representative isolate was chosen for de novo whole-genome assembly and for the alignment of sequence reads from the genomes of other phytoplasma isolates to detect polymorphisms. Statistical methods were applied to investigate the correlation between variations in disease symptoms of infected maize plants and MBSP sequence polymorphisms. Key Results: MBSP isolates contributed consistently to organ proliferation symptoms and maize genotype to leaf necrosis, reddening and yellowing of infected maize plants. The symptom differences are associated with polymorphisms in a phase-variable lipoprotein, which is a candidate effector, and an ATP-dependent lipoprotein ABC export protein, whereas no polymorphisms were observed in other candidate effector genes. Lipoproteins and ABC export proteins activate host defence responses, regulate pathogen attachment to host cells and activate effector secretion systems in other pathogens. Conclusions: Polymorphisms in two putative virulence genes among MBSP isolates from maize-growing regions in South-east Brazil are associated with variations in organ proliferation symptoms of MBSP-infected maize plants.