Quantitative PCR for Tracking the Megaplasmid-Borne Biodegradation Potential of a Model Sphingomonad

Hartmann, Erica M.; Badalamenti, Jonathan P.; Krajmalnik‐Brown, Rosa; Halden, Rolf U.

doi:10.1128/aem.00715-12

Cited by 9 publications

(5 citation statements)

References 23 publications

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“…The chromids and/or megaplasmids whose copy numbers have been examined in the family Rhizobiaceae (48)(49)(50) and the genera Burkholderia (51) have a copy number approximately equal to that of the chromosome. Similarly, the copy numbers of the large plasmids of Thermus thermophilus (52) and Sphingomonas wittichii (53) are similar to those of the chromosomes. However, the copy number of the chromid of the fast-replicating organism V. cholerae can actually be lower than that of the chromosome depending on the growth medium (54).…”

Section: Replication and Segregation Dynamics In Multipartite Genomesmentioning

confidence: 70%

The Divided Bacterial Genome: Structure, Function, and Evolution

diCenzo

Finan

2017

Microbiol Mol Biol Rev

202

262

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Approximately 10% of bacterial genomes are split between two or more large DNA fragments, a genome architecture referred to as a multipartite genome. This multipartite organization is found in many important organisms, including plant symbionts, such as the nitrogen-fixing rhizobia, and plant, animal, and human pathogens, including the genera ,, and . The availability of many complete bacterial genome sequences means that we can now examine on a broad scale the characteristics of the different types of DNA molecules in a genome. Recent work has begun to shed light on the unique properties of each class of replicon, the unique functional role of chromosomal and nonchromosomal DNA molecules, and how the exploitation of novel niches may have driven the evolution of the multipartite genome. The aims of this review are to (i) outline the literature regarding bacterial genomes that are divided into multiple fragments, (ii) provide a meta-analysis of completed bacterial genomes from 1,708 species as a way of reviewing the abundant information present in these genome sequences, and (iii) provide an encompassing model to explain the evolution and function of the multipartite genome structure. This review covers, among other topics, salient genome terminology; mechanisms of multipartite genome formation; the phylogenetic distribution of multipartite genomes; how each part of a genome differs with respect to genomic signatures, genetic variability, and gene functional annotation; how each DNA molecule may interact; as well as the costs and benefits of this genome structure.

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Section: Replication and Segregation Dynamics In Multipartite Genomesmentioning

confidence: 70%

The Divided Bacterial Genome: Structure, Function, and Evolution

diCenzo

Finan

2017

Microbiol Mol Biol Rev

202

262

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“…When utilizing S. wittichii RW1 as a bioremediation agent, it may be possible to induce the expression of the dioxin degradation pathway using acetate. Induction of the dioxin degradation pathway has not been observed when S. wittichii RW1 is grown on glucose or rich medium [ 22 ], and growth in a complex environmental medium (landfill leachate) was correlated with a decrease in copy number of the gene encoding the dioxin dioxygenase alpha subunit [ 37 ]. Previous studies using S. wittichii RW1 to transform chlorinated dioxins in soil or fly ash have observed a progressive decrease in degradative activity [ 5 ] or viable cells [ 38 , 39 ], respectively.…”

Section: Discussionmentioning

confidence: 99%

Proteomic Profiling of the Dioxin-Degrading BacteriumSphingomonas wittichiiRW1

Colquhoun

Hartmann

Halden

2012

Journal of Biomedicine and Biotechnology

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Sphingomonas wittichii RW1 is a bacterium of interest due to its ability to degrade polychlorinated dioxins, which represent priority pollutants in the USA and worldwide. Although its genome has been fully sequenced, many questions exist regarding changes in protein expression of S. wittichii RW1 in response to dioxin metabolism. We used difference gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to identify proteomic changes induced by growth on dibenzofuran, a surrogate for dioxin, as compared to acetate. Approximately 10% of the entire putative proteome of RW1 could be observed. Several components of the dioxin and dibenzofuran degradation pathway were shown to be upregulated, thereby highlighting the utility of using proteomic analyses for studying bioremediation agents. This is the first global protein analysis of a microorganism capable of utilizing the carbon backbone of both polychlorinated dioxins and dibenzofurans as the sole source for carbon and energy.

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“…A number of PCR primers are reported that can be used to probe samples for aromatic ring-hydroxylating dioxygenase genes (ARDHs) (Iwai et al, 2011b), including a quantitative PCR primer specific to the dxnA1 in Sphingomonas wittichii str. RW1, which is the only well-characterized dioxin degrader (Hartmann et al, 2012). However, no previously published primer set meeting the requirements for amplicon sequencing solely targets dioxygenases active toward dioxins (Iwai et al, 2011b).…”

Section: Aromatic Hydrocarbon-degrading Dxna Dbfa1 and Caraa Genesmentioning

confidence: 99%

Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter

Penton¹,

Johnson²,

Quensen³

et al. 2013

Front. Microbiol.

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Targeting sequencing to genes involved in key environmental processes, i.e., ecofunctional genes, provides an opportunity to sample nature's gene guilds to greater depth and help link community structure to process-level outcomes. Vastly different approaches have been implemented for sequence processing and, ultimately, for taxonomic placement of these gene reads. The overall quality of next generation sequence analysis of functional genes is dependent on multiple steps and assumptions of unknown diversity. To illustrate current issues surrounding amplicon read processing we provide examples for three ecofunctional gene groups. A combination of in silico, environmental and cultured strain sequences was used to test new primers targeting the dioxin and dibenzofuran degrading genes dxnA1, dbfA1, and carAa. The majority of obtained environmental sequences were classified into novel sequence clusters, illustrating the discovery value of the approach. For the nitrite reductase step in denitrification, the well-known nirK primers exhibited deficiencies in reference database coverage, illustrating the need to refine primer-binding sites and/or to design multiple primers, while nirS primers exhibited bias against five phyla. Amino acid-based OTU clustering of these two N-cycle genes from soil samples yielded only 114 unique nirK and 45 unique nirS genus-level groupings, likely a reflection of constricted primer coverage. Finally, supervised and non-supervised OTU analysis methods were compared using the nifH gene of nitrogen fixation, with generally similar outcomes, but the clustering (non-supervised) method yielded higher diversity estimates and stronger site-based differences. High throughput amplicon sequencing can provide inexpensive and rapid access to nature's related sequences by circumventing the culturing barrier, but each unique gene requires individual considerations in terms of primer design and sequence processing and classification.

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Quantitative PCR for Tracking the Megaplasmid-Borne Biodegradation Potential of a Model Sphingomonad

Cited by 9 publications

References 23 publications

The Divided Bacterial Genome: Structure, Function, and Evolution

The Divided Bacterial Genome: Structure, Function, and Evolution

Proteomic Profiling of the Dioxin-Degrading BacteriumSphingomonas wittichiiRW1

Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter

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