Partnership of Arthrobacter and Pimelobacter in Aerobic Degradation of Sulfadiazine Revealed by Metagenomics Analysis and Isolation

In this study, metagenomic analyses were combined with cultivation-based techniques as a nested approach to identify functionally significant bacteria for sulfadiazine biodegradation within enrichment communities. The metagenomic investigations indicated that our previously isolated sulfadiazine degrader, Arthrobacter sp. D2, and another Pimelobacter bacterium concomitantly occurred as most abundant members in the community of an enrichment culture that performed complete sulfadiazine mineralization for over two years. Responses of the enriched populations to sole carbon source alternation further suggested the ability of this Pimelobacter member to grow on 2-amino­pyrimidine, the most prominent intermediate metabolite of sulfadiazine. Taking advantage of this propensity, additional cultivation procedures have enabled the successful isolation of Pimelobacter sp. LG209, whose genomic sequences exactly matched that of the dominant Pimelobacter bacterium in the sulfadiazine enrichment culture. Integration of metagenomic investigations with the physiological characteristics of the isolates conclusively demonstrated that the sulfadiazine mineralization in a long-running enrichment culture was prominently mediated by primary sulfadiazine-degrading specialist strain Arthrobacter sp. D2 in association with the 2-amino­pyrimidine-degrading partner strain Pimelobacter sp. LG209. Here, we provided the first mechanistic insight into microbial interactions in steady sulfadiazine mineralization processes, which will help develop appropriate bioremediation strategies for sulfadiazine-contaminated hotspot sites

Genome Reconstruction and Gene Expression of “<i>Candidatus</i> Accumulibacter phosphatis” Clade IB Performing Biological Phosphorus Removal

We report the first integrated metatranscriptomic and metagenomic analysis of enhanced biological phosphorus removal (EBPR) sludge. A draft genome of <i>Candidatus</i> Accumulibacter spp. strain HKU-1, a member of Clade IB, was retrieved. It was estimated to be ∼90% complete and shared average nucleotide identities of 83% and 88% with the finished genome CAP IIA UW-1 and the draft genome CAP IA UW-2, respectively. Different from CAP IIA UW-1, the phosphotransferase (<i>pap</i>) in polyphosphate metabolism and <i>V-ATPase</i> in orthophosphate transport were absent from CAP IB HKU-1. Additionally, unlike CAP IA UW-2, CAP IB HKU-1 carried the genes for carbon fixation and nitrogen fixation. Despite these differences, the key genes required for acetate uptake, glycolysis and polyhydroxyalkanoate (PHA) synthesis were conserved in all these Accumulibacter genomes. The preliminary metatranscriptomic results revealed that the most significantly up-regulated genes of CAP IB HKU-1 from the anaerobic to the aerobic phase were responsible for assimilatory sulfate reduction, genetic information processing and phosphorus absorption, while the down-regulated genes were related to N₂O reduction, PHA synthesis and acetyl-CoA formation. This study yielded another important Accumulibacter genome, revealed the functional difference within the Accumulibacter Type I, and uncovered the genetic responses to EBPR stimuli at a higher resolution

Aerobic Degradation of Sulfadiazine by Arthrobacter spp.: Kinetics, Pathways, and Genomic Characterization

Two aerobic sulfadiazine (SDZ) degrading bacterial strains, D2 and D4, affiliated with the genus Arthrobacter, were isolated from SDZ-enriched activated sludge. The degradation of SDZ by the two isolates followed first-order decay kinetics. The half-life time of complete SDZ degradation was 11.3 h for strain D2 and 46.4 h for strain D4. Degradation kinetic changed from nongrowth to growth-linked when glucose was introduced as the cosubstrate, and accelerated biodegradation rate was observed after the adaption period. Both isolates could degrade SDZ into 12 biodegradation products via 3 parallel pathways, of which 2-amino-4-hydroxypyrimidine was detected as the principal intermediate product toward the pyrimidine ring cleavage. Compared with five Arthrobacter strains reported previously, D2 and D4 were the only Arthrobacter strains which could degrade SDZ as the sole carbon source. The draft genomes of D2 and D4, with the same completeness of 99.7%, were compared to other genomes of related species. Overall, these two isolates shared high genomic similarities with the <i>s</i>-triazine-degrading Arthrobacter sp. AK-YN10 and the sulfonamide-degrading bacteria Microbacterium sp. C448. In addition, the two genomes contained a few significant regions of difference which may carry the functional genes involved in sulfonamide degradation

Characteristics of assembled scaffolds and the retrieved CAP IIC HKU-2 draft genome.

<p>Characteristics of assembled scaffolds and the retrieved CAP IIC HKU-2 draft genome.</p

Phosphorus removal and sludge sampling for 16S rRNA gene pyrosequencing from the SBR performing EBPR.

<p>The pH of solution in the SBR was maintained at 7.2 ± 0.1 except at 60 d when accidentally overdosed acidic solution to decrease pH to 6.0 for around 20 h.</p

Venn diagram of conserved and unique genes for four Accumulibacter genomes of Clade IIC.

<p>Venn diagram of conserved and unique genes for four Accumulibacter genomes of Clade IIC.</p

Exploring the Shift in Structure and Function of Microbial Communities Performing Biological Phosphorus Removal

<div><p>A sequencing batch reactor fed mainly by acetate was operated to perform enhanced biological phosphorus removal (EBPR). A short-term pH shock from 7.0 to 6.0 led to a complete loss of phosphate-removing capability and a drastic change of microbial communities. 16S rRNA gene pyrosequencing showed that large proportions of glycogen accumulating organisms (GAOs) (accounted for 16% of bacteria) bloomed, including <i>Candidatus</i> Competibacter phosphatis and <i>Defluviicoccus</i>-related tetrad-forming organism, causing deteriorated EBPR performance. The EBPR performance recovered with time and the dominant <i>Candidatus</i> Accumulibacter (Accumulibacter) clades shifted from Clade IIC to IIA while GAOs populations shrank significantly. The Accumulibacter population variation provided a good opportunity for genome binning using a bi-dimensional coverage method, and a genome of Accumulibacter Clade IIC was well retrieved with over 90% completeness. Comparative genomic analysis demonstrated that Accumulibacter clades had different abilities in nitrogen metabolism and carbon fixation, which shed light on enriching different Accumulibacter populations selectively.</p></div

Maximum likelihood phylogenetic tree of Accumulibacter <i>ppk1</i> gene sequences.

<p>The <i>ppk1</i> genes of the reconstructed Accumulibacter genomes are indicated in green, while those from clone library of sludges A and C are colored in orange. Seven and nine partial <i>ppk1</i> gene sequences with 99% identity were obtained from sludge A and C respectively. Reference sequences attached with their accession numbers are extracted from NCBI database. Node labels refer to bootstrap support values and <i>Rhodocyclus tenuis ppk1</i> gene is employed as the outgroup sequence.</p

The ANI and orthologous genes shared by each pair of Accumulibacter genomes.

<p>The ANI and orthologous genes shared by each pair of Accumulibacter genomes.</p

Relative abundances of <i>ppk1</i> genes of different clades in the microbial communities.

<p>The abundance of Accumulibacter was calculated according to the copy numbers of Accumulibacter and bacterial 16S rRNA genes by qPCR analysis. Meanwhile the 2 copies of <i>rrn</i> operon in CAP IIA UW-1 and 4 copies of <i>rrn</i> operon in the available bacterial finished genomes have been taken into account. The proportions of different <i>ppk1</i> genes in one sample was estimated by the copy numbers obtained from the qPCR assay using primer sets targeting <i>ppk1</i> genes of specific clades.</p