Comparative genome analysis reveals genetic adaptation to versatile environmental conditions and importance of biofilm lifestyle in Comamonas testosteroni.

Comamonas testosteroni is an important environmental bacterium capable of degrading a variety of toxic aromatic pollutants and has been demonstrated to be a promising biocatalyst for environmental decontamination. This organism is often found to be among the primary surface colonizers in various natural and engineered ecosystems, suggesting an extraordinary capability of this organism in environmental adaptation and biofilm formation. The goal of this study was to gain genetic insights into the adaption of C. testosteroni to versatile environments and the importance of a biofilm lifestyle. Specifically, a draft genome of C. testosteroni I2 was obtained. The draft genome is 5,778,710 bp in length and comprises 110 contigs. The average G+C content was 61.88 %. A total of 5365 genes with 5263 protein-coding genes were predicted, whereas 4324 (80.60 % of total genes) protein-encoding genes were associated with predicted functions. The catabolic genes responsible for biodegradation of steroid and other aromatic compounds on draft genome were identified. Plasmid pI2 was found to encode a complete pathway for aniline degradation and a partial catabolic pathway for chloroaniline. This organism was found to be equipped with a sophisticated signaling system which helps it find ideal niches and switch between planktonic and biofilm lifestyles. A large number of putative multi-drug-resistant genes coding for abundant outer membrane transporters, chaperones, and heat shock proteins for the protection of cellular function were identified in the genome of strain I2. In addition, the genome of strain I2 was predicted to encode several proteins involved in producing, secreting, and uptaking siderophores under iron-limiting conditions. The genome of strain I2 contains a number of genes responsible for the synthesis and secretion of exopolysaccharides, an extracellular component essential for biofilm formation. Overall, our results reveal the genomic features underlying the adaption of C. testosteroni to versatile environments and highlighting the importance of its biofilm lifestyle.

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.

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.

Controlling anammox speciation and biofilm attachment strategy using N-biotransformation intermediates and organic carbon levels.

Conventional nitrogen removal in wastewater treatment requires a high oxygen and energy input. Anaerobic ammonium oxidation (anammox), the single-step conversion of ammonium and nitrite to nitrogen gas, is a more energy and cost effective alternative applied extensively to sidestream wastewater treatment. It would also be a mainstream treatment option if species diversity and physiology were better understood. Anammox bacteria were enriched up to 80%, 90% and 50% relative abundance, from a single inoculum, under standard enrichment conditions with either stepwise-nitrite and ammonia concentration increases (R1), nitric oxide supplementation (R2), or complex organic carbon from mainstream wastewater (R3), respectively. Candidatus Brocadia caroliniensis predominated in all reactors, but a shift towards Ca. Brocadia sinica occurred at ammonium and nitrite concentrations > 270 mg NH4-N L-1 and 340 mg NO2-N L-1 respectively. With NO present, heterotrophic growth was inhibited, and Ca. Jettenia coexisted with Ca. B. caroliniensis before diminishing as nitrite increased to 160 mg NO2-N L-1. Organic carbon supplementation led to the emergence of heterotrophic communities that coevolved with Ca. B. caroliniensis. Ca. B. caroliniensis and Ca. Jettenia preferentially formed biofilms on surfaces, whereas Ca. Brocadia sinica formed granules in suspension. Our results indicate that multiple anammox bacteria species co-exist and occupy sub-niches in anammox reactors, and that the dominant population can be reversibly shifted by, for example, changing nitrogen load (i.e. high nitrite concentration favors Ca. Brocadia caroliniensis). Speciation has implications for wastewater process design, where the optimum cell immobilization strategy (i.e. carriers vs granules) depends on which species dominates.

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.

Tropical-based EBPR process: The long-term stability, microbial community and its response towards temperature stress.

Temperature is known to influence the operational efficiency of enhanced biological phosphorus removal (EBPR) systems. This study investigated the impact of thermal stress above 30°C on the properties of an EBPR community established with tropical inoculum. The results confirmed the stability of the 30°C EBPR system with high P-removal efficiency over 210 days. Accumulibacter was abundant in the community. When the EBPR sludge was subjected to a sudden temperature increase to 35°C under multiple cycles of anaerobic-aerobic phases, each lasting 4 h, high P-removal was maintained over 2 days, before gradually failing when the Competibacter appeared to outcompete Accumulibacter. These data suggested that the EBPR capacity is robust when subjected to occasional thermal stress. However, it could not be maintained even for a short time under temperature stress at 40°C. Thus, the threshold temperature for tropical EBPR failure is between 35°C and 40°C. PRACTITIONER POINTS: EBPR was stably maintained at 30°C with Accumulibacter being dominant. Good EBPR activities persisted for a short period at 35°C. EBPR was deteriorated at 40°C. The threshold temperature for tropical EBPR failure is between 35°C and 40°C.

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.

Annotated bacterial chromosomes from frame-shift-corrected long-read metagenomic data.

BACKGROUND: Short-read sequencing technologies have long been the work-horse of microbiome analysis. Continuing technological advances are making the application of long-read sequencing to metagenomic samples increasingly feasible. RESULTS: We demonstrate that whole bacterial chromosomes can be obtained from an enriched community, by application of MinION sequencing to a sample from an EBPR bioreactor, producing 6 Gb of sequence that assembles into multiple closed bacterial chromosomes. We provide a simple pipeline for processing such data, which includes a new approach to correcting erroneous frame-shifts. CONCLUSIONS: Advances in long-read sequencing technology and corresponding algorithms will allow the routine extraction of whole chromosomes from environmental samples, providing a more detailed picture of individual members of a microbiome.

Draft Genome Sequence of a "Candidatus Brocadia" Bacterium Enriched from Activated Sludge Collected in a Tropical Climate.

Here, we present the draft genome sequence of an anaerobic ammonium-oxidizing bacterium (AnAOB), "Candidatus Brocadia," which was enriched in an anammox reactor. A 3.2-Mb genome sequence comprising 168 contigs was assembled, in which 2,765 protein-coding genes, 47 tRNAs, and one each of 5S, 16S, and 23S rRNAs were annotated. No evidence for the presence of a nitric oxide-forming nitrite reductase was found.

Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions.

Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions.

Draft Genome Sequence of Klebsiella pneumoniae Strain KP-1.

Klebsiella pneumoniae is ubiquitous in the environment and is a member of a three-species biofilm model. We compared the genome sequence of an environmental isolate, K. pneumoniae strain KP-1, to those of two clinical strains (NTUH-K2044 and MGH 78578). KP-1 possesses strain-specific prophage sequences that distinguish it from the clinical strains.

A database for medicinal plants used in treatment of asthma.

UNLABELLED: The knowledge of most plants used in the treatment of asthma, the plant part which is effective in treatment is confined to very few persons who are engaged in folklore medicine. However, this form of medicine is not very popular. Therefore, it is of considerable interest to ethno-botanical community to understand the plants and the parts used for treatment. Here, we describe AsthmaPlantBase, a database containing information of medicinal plants for treatment of asthma. AVAILABILITY: http://www.asthmaplants.com.