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.

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.