Shaomei He

Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities

Enhanced biological phosphorus removal (EBPR) is one of the best-studied microbially mediated industrial processes because of its ecological and economic relevance. Despite this, it is not well understood at the metabolic level. Here we present a metagenomic analysis of two lab-scale EBPR sludges dominated by the uncultured bacterium, “Candidatus Accumulibacter phosphatis.” The analysis sheds light on several controversies in EBPR metabolic models and provides hypotheses explaining the dominance of A. phosphatis in this habitat, its lifestyle outside EBPR and probable cultivation requirements. Comparison of the same species from different EBPR sludges highlights recent evolutionary dynamics in the A. phosphatis genome that could be linked to mechanisms for environmental adaptation. In spite of an apparent lack of phylogenetic overlap in the flanking communities of the two sludges studied, common functional themes were found, at least one of them complementary to the inferred metabolism of the dominant organism. The present study provides a much needed blueprint for a systems-level understanding of EBPR and illustrates that metagenomics enables detailed, often novel, insights into even well-studied biological systems.

“Candidatus Accumulibacter” Population Structure in Enhanced Biological Phosphorus Removal Sludges as Revealed by Polyphosphate Kinase Genes

ABSTRACT We investigated the fine-scale population structure of the “Candidatus Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of “Candidatus Accumulibacter” 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “Candidatus Accumulibacter” lineage. Sequences from at least five clades of “Candidatus Accumulibacter” were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using “Candidatus Accumulibacter”-specific 16S rRNA and “Candidatus Accumulibacter” clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “Candidatus Accumulibacter” lineage and the relative distributions and abundances of the five “Candidatus Accumulibacter” clades. The qPCR-based estimation of the total “Candidatus Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined “Candidatus Accumulibacter” clades. The relative distributions of “Candidatus Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “Candidatus Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.

Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion, as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.

Comparative Metagenomic and Metatranscriptomic Analysis of Hindgut Paunch Microbiota in Wood- and Dung-Feeding Higher Termites

Termites effectively feed on many types of lignocellulose assisted by their gut microbial symbionts. To better understand the microbial decomposition of biomass with varied chemical profiles, it is important to determine whether termites harbor different microbial symbionts with specialized functionalities geared toward different feeding regimens. In this study, we compared the microbiota in the hindgut paunch of Amitermes wheeleri collected from cow dung and Nasutitermes corniger feeding on sound wood by 16S rRNA pyrotag, comparative metagenomic and metatranscriptomic analyses. We found that Firmicutes and Spirochaetes were the most abundant phyla in A. wheeleri, in contrast to N. corniger where Spirochaetes and Fibrobacteres dominated. Despite this community divergence, a convergence was observed for functions essential to termite biology including hydrolytic enzymes, homoacetogenesis and cell motility and chemotaxis. Overrepresented functions in A. wheeleri relative to N. corniger microbiota included hemicellulose breakdown and fixed-nitrogen utilization. By contrast, glycoside hydrolases attacking celluloses and nitrogen fixation genes were overrepresented in N. corniger microbiota. These observations are consistent with dietary differences in carbohydrate composition and nutrient contents, but may also reflect the phylogenetic difference between the hosts.

Comparative genomics of two 'Candidatus Accumulibacter' clades performing biological phosphorus removal

Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80–90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience.

‘ Candidatus Accumulibacter’ gene expression in response to dynamic EBPR conditions

Enhanced biological phosphorus removal (EBPR) activated sludge communities enriched in ‘Candidatus Accumulibacter’ relatives are widely used in wastewater treatment, but much remains to be learned about molecular-level controls on the EBPR process. The expression of genes found in the carbon and polyphosphate metabolic pathways in Accumulibacter was investigated using reverse transcription quantitative PCR. During a normal anaerobic/aerobic EBPR cycle, gene expression exhibited a dynamic change in response to external acetate, oxygen, phosphate concentrations and probably internal chemical pools. Anaerobic acetate addition induced expression of genes associated with the methylmalonyl-CoA pathway enabling the split mode of the tricarboxylic acid (TCA) cycle. Components of the full TCA cycle were induced after the switch to aerobic conditions. The induction of a key gene in the glyoxylate shunt pathway was observed under both anaerobic and aerobic conditions, with a higher induction by aeration. Polyphosphate kinase 1 from Accumulibacter was expressed, but did not appear to be regulated by phosphate limitation. To understand how Accumulibacter responds to disturbed electron donor and acceptor conditions, we perturbed the process by adding acetate aerobically. When high concentrations of oxygen were present simultaneously with acetate, phosphate-release was almost completely inhibited, and polyphosphate kinase 1 transcript abundance decreased. Genes associated with the methylmalonyl-CoA pathway were repressed and genes associated with the aerobic TCA cycle exhibited higher expression under this perturbation, suggesting that more acetyl-CoA was metabolized through the TCA cycle. These findings suggest that several genes involved in EBPR are tightly regulated at the transcriptional level.

Microbiology of ‘Candidatus Accumulibacter’ in activated sludge

‘Candidatus Accumulibacter’ is a biotechnologically important bacterial group that can accumulate large amounts of intracellular polyphosphate, contributing to biological phosphorus removal in wastewater treatment. Since its first molecular identification more than a decade ago, this bacterial group has drawn significant research attention due to its high abundance in many biological phosphorus removal systems. In the past 6 years, our understanding of Accumulibacter microbiology and ecophysiology has advanced rapidly, largely owing to genomic information obtained through shotgun metagenomic sequencing efforts. In this review, we focus on the metabolism, physiology, fine‐scale population structure and ecological distribution of Accumulibacter, aiming to integrate the information learned so far and to present a more complete picture of the microbiology of this important bacterial group.

Phage encoded H-NS: a potential achilles heel in the bacterial defence system.

The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR), offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP), led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS) protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1), and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race.

Bacterial community and "Candidatus Accumulibacter" population dynamics in laboratory-scale enhanced biological phosphorus removal reactors.

ABSTRACT “Candidatus Accumulibacter” and total bacterial community dynamics were studied in two lab-scale enhanced biological phosphorus removal (EBPR) reactors by using a community fingerprint technique, automated ribosomal intergenic spacer analysis (ARISA). We first evaluated the quantitative capability of ARISA compared to quantitative real-time PCR (qPCR). ARISA and qPCR provided comparable relative quantification of the two dominant “Ca. Accumulibacter” clades (IA and IIA) detected in our reactors. The quantification of total “Ca. Accumulibacter” 16S rRNA genes relative to that from the total bacterial community was highly correlated, with ARISA systematically underestimating “Ca. Accumulibacter” abundance, probably due to the different normalization techniques applied. During 6 months of normal (undisturbed) operation, the distribution of the two clades within the total “Ca. Accumulibacter” population was quite stable in one reactor while comparatively dynamic in the other reactor. However, the variance in the clade distribution did not appear to affect reactor performance. Instead, good EBPR activity was positively associated with the abundance of total “Ca. Accumulibacter.” Therefore, we concluded that the different clades in the system provided functional redundancy. We disturbed the reactor operation by adding nitrate together with acetate feeding in the anaerobic phase to reach initial reactor concentrations of 10 mg/liter NO3-N for 35 days. The reactor performance deteriorated with a concomitant decrease in the total “Ca. Accumulibacter” population, suggesting that a population shift was the cause of performance upset after a long exposure to nitrate in the anaerobic phase.

Ecophysiology of Freshwater Verrucomicrobia Inferred from Metagenome-Assembled Genomes

Freshwater Verrucomicrobia spp. are cosmopolitan in lakes and rivers, and yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia spp. are restricted to one subdivision of this phylum. Here, we greatly expanded the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent the first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia to be potential (poly)saccharide degraders and suggested their adaptation to carbon sources of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called “Planctomycete-specific” cytochrome c-encoding genes and identified their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology, and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling. ABSTRACT Microbes are critical in carbon and nutrient cycling in freshwater ecosystems. Members of the Verrucomicrobia are ubiquitous in such systems, and yet their roles and ecophysiology are not well understood. In this study, we recovered 19 Verrucomicrobia draft genomes by sequencing 184 time-series metagenomes from a eutrophic lake and a humic bog that differ in carbon source and nutrient availabilities. These genomes span four of the seven previously defined Verrucomicrobia subdivisions and greatly expand knowledge of the genomic diversity of freshwater Verrucomicrobia. Genome analysis revealed their potential role as (poly)saccharide degraders in freshwater, uncovered interesting genomic features for this lifestyle, and suggested their adaptation to nutrient availabilities in their environments. Verrucomicrobia populations differ significantly between the two lakes in glycoside hydrolase gene abundance and functional profiles, reflecting the autochthonous and terrestrially derived allochthonous carbon sources of the two ecosystems, respectively. Interestingly, a number of genomes recovered from the bog contained gene clusters that potentially encode a novel porin-multiheme cytochrome c complex and might be involved in extracellular electron transfer in the anoxic humus-rich environment. Notably, most epilimnion genomes have large numbers of so-called “Planctomycete-specific” cytochrome c-encoding genes, which exhibited distribution patterns nearly opposite to those seen with glycoside hydrolase genes, probably associated with the different levels of environmental oxygen availability and carbohydrate complexity between lakes/layers. Overall, the recovered genomes represent a major step toward understanding the role, ecophysiology, and distribution of Verrucomicrobia in freshwater. IMPORTANCE Freshwater Verrucomicrobia spp. are cosmopolitan in lakes and rivers, and yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia spp. are restricted to one subdivision of this phylum. Here, we greatly expanded the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent the first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia to be potential (poly)saccharide degraders and suggested their adaptation to carbon sources of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called “Planctomycete-specific” cytochrome c-encoding genes and identified their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology, and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling.