For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence, DNA methylation is characterized in the process of sequencing. PacBio® sequencing has also been applied to microbial transcriptomes. Long reads enable sequencing of full-length cDNAs allowing for identification of complete gene and operon sequences without the need for transcript assembly. We will highlight several examples where these capabilities have been leveraged in the areas of industrial microbiology, including biocommodities, biofuels, bioremediation, new bacteria with potential commercial applications, antibiotic discovery, and livestock/plant microbiome interactions.
Despite apparent carbon limitation, anoxic deep subsurface brines at the Soudan Underground Iron Mine harbor active microbial communities. To characterize these assemblages, we performed shotgun metagenomics of native and enriched samples. Following enrichment on poised electrodes and long read sequencing, we recovered from the metagenome the closed, circular genome of a novel Desulfuromonas sp. with remarkable genomic features that were not fully resolved by short read assembly alone. This organism was essentially absent in unenriched Soudan communities, indicating that electrodes are highly selective for putative metal reducers. Native community metagenomes suggest that carbon cycling is driven by methyl-C1 metabolism, in particular methylotrophic methanogenesis. Our results highlight the promising potential for long reads in metagenomic surveys of low-diversity environments.
N,N-Dimethylformamide (DMF) is one of the most common xenobiotic chemicals, and it can be easily emitted into the environment, where it causes harm to human beings. Herein, an efficient DMF-degrading strain, DM1, was isolated and identified as Methylobacterium sp. This strain can use DMF as the sole source of carbon and nitrogen. Whole-genome sequencing of strain DM1 revealed that it has a 5.66-Mbp chromosome and a 200-kbp megaplasmid. The plasmid pLVM1 specifically harbors the genes essential for the initial steps of DMF degradation, and the chromosome carries the genes facilitating subsequent methylotrophic metabolism. Through analysis of the transcriptome sequencing data, the complete mineralization pathway and redundant gene clusters of DMF degradation were elucidated. The dimethylformamidase (DMFase) gene was heterologously expressed, and DMFase was purified and characterized. Plasmid pLVM1 is catabolically crucial for DMF utilization, as evidenced by the phenotype identification of the plasmid-free strain. This study systematically elucidates the molecular mechanisms of DMF degradation by MethylobacteriumIMPORTANCE DMF is a hazardous pollutant that has been used in the chemical industry, pharmaceutical manufacturing, and agriculture. Biodegradation as a method for removing DMF has received increasing attention. Here, we identified an efficient DMF degrader, Methylobacterium sp. strain DM1, and characterized the complete DMF mineralization pathway and enzymatic properties of DMFase in this strain. This study provides insights into the molecular mechanisms and evolutionary advantage of DMF degradation facilitated by plasmid pLVM1 and redundant genes in strain DM1, suggesting the emergence of new ecotypes of Methylobacterium.Copyright © 2019 American Society for Microbiology.
Whole genome sequencing of a novel, dichloromethane-fermenting Peptococcaceae from an enrichment culture
Bacteria capable of dechlorinating the toxic environmental contaminant dichloromethane (DCM, CHt2Cl2) are of great interest for potential bioremediation applications. A novel, strictly anaerobic, DCM-fermenting bacterium, “DCMF”, was enriched from organochlorine-contaminated groundwater near Botany Bay, Australia. The enrichment culture was maintained in minimal, mineral salt medium amended with dichloromethane as the sole energy source. PacBio whole genome SMRTtextsuperscriptTM sequencing of DCMF allowed textitde novo, gap-free assembly despite the presence of cohabiting organisms in the culture. Illumina sequencing reads were utilised to correct minor indels. The single, circularised 6.44 Mb chromosome was annotated with the IMG pipeline and contains 5,773 predicted protein-coding genes. Based on 16S rRNA gene and predicted proteome phylogeny, the organism appears to be a novel member of the textitPeptococcaceae family. The DCMF genome is large in comparison to known DCM-fermenting bacteria and includes 96 predicted methylamine methyltransferases, which may provide clues to the basis of its DCM metabolism. Full annotation has been provided in a custom genome browser and search tool, in addition to multiple sequence alignments and phylogenetic trees for every predicted protein, available at http://www.slimsuite.unsw.edu.au/research/dcmf/.
Carbaryl was a widely used pesticide in the agriculture industry. The toxicity against non-target organisms and the environmental pollution it caused became the focus of public concern. However, the microbial mechanism of carbaryl degradation was not fully investigated. In the study, we reported the complete genome of the carbaryl-degrading Pseudomonas putida strain XWY-1, which consists of a chromosome (5.9 Mbp) and a plasmid (0.4 Mbp). The carbaryl degradation genes are located on the plasmid. The study on the genome will facilitate to further elucidate the carbaryl degradation and advance the potential biotechnological applications of P. putida strain XWY-1.
Complete genome sequence of Paracoccus denitrificans ATCC 19367 and its denitrification characteristics.
Studies show that Paracoccus denitrificans can denitrify nitrogen sources under aerobic conditions. However, the lack of data on its genome sequence has restricted molecular studies and practical applications. In this study, the complete genome of P. denitrificans ATCC 19367 was sequenced and its nitrogen metabolism properties were characterized. The size of the whole genome is 5?242?327 bp, with two chromosomes and one plasmid. The average G + C content is 66.8%, and it contains 5308 protein-coding genes, 54 tRNA genes, and nine rRNA operons. Among the protein-coding genes, 71.35% could be assigned to the Gene Ontology (GO) pathway, 86.66% to the Clusters of Orthologous Groups (COG) pathway, and 50.57% to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Comparative genome analysis between P. denitrificans ATCC 19367 and P. denitrificans PD1222 revealed that there are 428 genes specific to ATCC 19367 and 4738 core genes. Furthermore, the expression of genes related to denitrification, biofilm formation, and nitrogen metabolism (nar, nir, and nor) by P. denitrificans ATCC 19367 under aerobic conditions was affected by incubation time and shaking speed. This study elucidates the genomic background of P. denitrificans ATCC 19367 and suggests the possibility of controlling nitrogen pollution in the environment by using this bacterium.
Characterization of the genome of a Nocardia strain isolated from soils in the Qinghai-Tibetan Plateau that specifically degrades crude oil and of this biodegradation.
A strain of Nocardia isolated from crude oil-contaminated soils in the Qinghai-Tibetan Plateau degrades nearly all components of crude oil. This strain was identified as Nocardia soli Y48, and its growth conditions were determined. Complete genome sequencing showed that N. soli Y48 has a 7.3?Mb genome and many genes responsible for hydrocarbon degradation, biosurfactant synthesis, emulsification and other hydrocarbon degradation-related metabolisms. Analysis of the clusters of orthologous groups (COGs) and genomic islands (GIs) revealed that Y48 has undergone significant gene transfer events to adapt to changing environmental conditions (crude oil contamination). The structural features of the genome might provide a competitive edge for the survival of N. soli Y48 in oil-polluted environments and reflect the adaptation of coexisting bacteria to distinct nutritional niches.Copyright © 2018. Published by Elsevier Inc.
Whole Genome Sequencing and Comparative Genomics Analyses of Pandoraea sp. XY-2, a New Species Capable of Biodegrade Tetracycline.
Few bacteria are resistant to tetracycline and can even biodegrade tetracycline in the environment. In this study, we isolated a bacterium Pandoraea sp. XY-2, which could biodegrade 74% tetracycline at pH 7.0 and 30°C within 6 days. Thereafter, we determined the whole genome sequence of Pandoraea sp. XY-2 genome is a single circular chromosome of 5.06 Mb in size. Genomic annotation showed that two AA6 family members-encoding genes and nine glutathione S-transferase (GSTs)-encoding genes could be relevant to tetracycline biodegradation. In addition, the average nucleotide identities (ANI) analysis between the genomes of Pandoraea sp. XY-2 and other Pandoraea spp. revealed that Pandoraea sp. XY-2 belongs to a new species. Moreover, comparative genome analysis of 36 Pandoraea strains identified the pan and specific genes, numerous single nucleotide polymorphisms (SNPs), insertions, and deletion variations (InDels) and different syntenial relationships in the genome of Pandoraea sp. XY-2. Finally, the evolution and the origin analysis of genes related to tetracycline resistance revealed that the six tetA(48) genes and two specificgenes tetG and tetR in Pandoraea sp. XY-2 were acquired by horizontal gene transfer (HGT) events from sources related to Paraburkholderia, Burkholderia, Caballeronia, Salmonella, Vibrio, Proteobacteria, Pseudomonas, Acinetobacter, Flavimaricola, and some unidentified sources. As a new species, Pandoraea sp. XY-2 will be an excellent resource for the bioremediation of tetracycline-contaminated environment.
PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.
Biodegradation of nonylphenol during aerobic composting of sewage sludge under two intermittent aeration treatments in a full-scale plant.
The urbanization and industrialization of cities around the coastal region of the Bohai Sea have produced large amounts of sewage sludge from sewage treatment plants. Research on the biodegradation of nonylphenol (NP) and the influencing factors of such biodegradation during sewage sludge composting is important to control pollution caused by land application of sewage sludge. The present study investigated the effect of aeration on NP biodegradation and the microbe community during aerobic composting under two intermittent aeration treatments in a full-scale plant of sewage sludge, sawdust, and returned compost at a ratio of 6:3:1. The results showed that 65% of NP was biodegraded and that Bacillus was the dominant bacterial species in the mesophilic phase. The amount of NP biodegraded in the mesophilic phase was 68.3%, which accounted for 64.6% of the total amount of biodegraded NP. The amount of NP biodegraded under high-volume aeration was 19.6% higher than that under low-volume aeration. Bacillus was dominant for 60.9% of the composting period under high-volume aeration, compared to 22.7% dominance under low-volume aeration. In the thermophilic phase, high-volume aeration promoted the biodegradation of NP and Bacillus remained the dominant bacterial species. In the cooling and stable phases, the contents of NP underwent insignificant change while different dominant bacteria were observed in the two treatments. NP was mostly biodegraded by Bacillus, and the rate of biodegradation was significantly correlated with the abundance of Bacillus (r?=?0.63, p?0.05). Under aeration, Bacillus remained the dominant bacteria, especially in the thermal phase; this phenomenon possibly increased the biodegradation efficiency of NP. High-volume aeration accelerated the activity and prolonged the survival of Bacillus. The risk of organic pollution could be decreased prior to sewage sludge reuse in soil by adjusting the ventilation strategies of aerobic compost measurements. Copyright © 2018 Elsevier Ltd. All rights reserved.
Composition and pathogenic potential of a microbial bioremediation product used for crude oil degradation.
A microbial bioremediation product (MBP) used for large-scale oil degradation was investigated for microbial constituents and possible pathogenicity. Aerobic growth on various media yielded >108 colonies mL-1. Full-length 16S rDNA sequencing and fatty acid profiling from morphologically distinct colonies revealed =13 distinct genera. Full-length 16S rDNA library sequencing, by either Sanger or long-read PacBio technology, suggested that up to 21% of the MBP was composed of Arcobacter. Other high abundance microbial constituents (>6%) included the genera Proteus, Enterococcus, Dysgonomonas and several genera in the order Bacteroidales. The MBP was most susceptible to ciprofloxacin, doxycycline, gentamicin, and meropenam. MBP exposure of human HT29 and A549 cells caused significant cytotoxicity, and bacterial growth and adherence. An acellular MBP filtrate was also cytotoxic to HT29, but not A549. Both MBP and filtrate exposures elevated the neutrophil chemoattractant IL-8. In endotracheal murine exposures, bacterial pulmonary clearance was complete after one-week. Elevation of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-a, and chemokines KC and MCP-1 occurred between 2h and 48h post-exposure, followed by restoration to baseline levels at 96h. Cytokine/chemokine signalling was accompanied by elevated blood neutrophils and monocytes at 4h and 48h, respectively. Peripheral acute phase response markers were maximal at 24h. All indicators examined returned to baseline values by 168h. In contrast to HT29, but similar to A549 observations, MBP filtrate did not induce significant murine effects with the indicators examined. The results demonstrated the potentially complex nature of MBPs and transient immunological effects during exposure. Products containing microbes should be scrutinized for pathogenic components and subjected to characterisation and quality validation prior to commercial release.
Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene.
A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Adaptive strategies of Bacillus thuringiensis isolated from acid mine drainage site in Sabah, Malaysia.
The adaptive process in bacteria is driven by specific genetic elements which regulate phenotypic characteristics such as tolerance to high metal ion concentrations and the secretion of protective biofilms. Extreme environments such as those associated with heavy metal pollution and extremes of acidity offer opportunities to study the adaptive mechanisms of microorganisms. This study focused on the genome analysis of Bacillus thuringiensis (Bt MCMY1), a gram positive rod shaped bacterium isolated from an acid mine drainage site in Sabah, Malaysia by using a combination of Single Molecule Real Time DNA Sequencing, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The genome size of Bt MCMY1 was determined to be 5,458,152 bases which was encoded on a single chromosome. Analysis of the genome revealed genes associated with resistance to Copper, Mercury, Arsenic, Cobalt, Zinc, Cadmium and Aluminum. Evidence from SEM and FTIR indicated that the bacterial colonies form distinct films which bear the signature of polyhydroxyalkanoates (PHA) and this finding was supported by the genome data indicating the presence of a genetic pathway associated with the biosynthesis of PHAs. This is the first report of a Bacillus sp. isolated from an acid mine drainage site in Sabah, Malaysia and the genome sequence will provide insights into the manner in which B. thuringiensis adapts to acid mine drainage.
Biodegradation of decabromodiphenyl ether (BDE 209) by a newly isolated bacterium from an e-waste recycling area.
Polybrominated diphenyl ethers (PBDEs) have become widespread environmental pollutants all over the world. A newly isolated bacterium from an e-waste recycling area, Stenotrophomonas sp. strain WZN-1, can degrade decabromodiphenyl ether (BDE 209) effectively under aerobic conditions. Orthogonal test results showed that the optimum conditions for BDE 209 biodegradation were pH 5, 25 °C, 0.5% salinity, 150 mL minimal salt medium volume. Under the optimized condition, strain WZN-1 could degrade 55.15% of 65 µg/L BDE 209 under aerobic condition within 30 day incubation. Moreover, BDE 209 degradation kinetics was fitted to a first-order kinetics model. The biodegradation mechanism of BDE 209 by strain WZN-1 were supposed to be three possible metabolic pathways: debromination, hydroxylation, and ring opening processes. Four BDE 209 degradation genes, including one hydrolase, one dioxygenase and two dehalogenases, were identified based on the complete genome sequencing of strain WZN-1. The real-time qPCR demonstrated that the expression level of four identified genes were significantly induced by BDE 209, and they played an important role in the degradation process. This study is the first to demonstrate that the newly isolated Stenotrophomonas strain has an efficient BDE 209 degradation ability and would provide new insights for the microbial degradation of PBDEs.
Rhizospheric microbial communities are driven by Panax ginseng at different growth stages and biocontrol bacteria alleviates replanting mortality
The cultivation of Panax plants is hindered by replanting problems, which may be caused by plant-driven changes in the soil microbial community. Inoculation with microbial antagonists may efficiently alleviate replanting issues. Through high-throughput sequencing, this study revealed that bacterial diversity decreased, whereas fungal diversity increased, in the rhizosphere soils of adult ginseng plants at the root growth stage under different ages. Few microbial community, such as Luteolibacter, Cytophagaceae, Luteibacter, Sphingomonas, Sphingomonadaceae, and Zygomycota, were observed; the relative abundance of microorganisms, namely, Brevundimonas, Enterobacteriaceae, Pandoraea, Cantharellales, Dendryphion, Fusarium, and Chytridiomycota, increased in the soils of adult ginseng plants compared with those in the soils of 2-year-old seedlings. Bacillus subtilis 50-1, a microbial antagonist against the pathogenic Fusarium oxysporum, was isolated through a dual culture technique. These bacteria acted with a biocontrol efficacy of 67.8%. The ginseng death rate and Fusarium abundance decreased by 63.3% and 46.1%, respectively, after inoculation with B. subtilis 50-1. Data revealed that microecological degradation could result from ginseng-driven changes in rhizospheric microbial communities; these changes are associated with the different ages and developmental stages of ginseng plants. Biocontrol using microbial antagonists alleviated the replanting problem.