Draft Genome Sequences of Two Novel Sub-Antarctic
            <i>Williamsia</i>
            Species

Nahar, Akhikun; Baker, Anthony L.; Charleston, Michael A.; Bowman, John P.; Britz, ML

doi:10.1128/genomea.01047-17

Cited by 2 publications

(4 citation statements)

References 9 publications

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“…We were able to shotgun sequence six unclassified sub-Antarctic Corynebacteriales strains [27][28][29] after optimizing gDNA extraction using a method based on those described by Marmur [14,15]. This method was only successful when the strains were cultured in broths containing concentrations of glycine that were highly growth-inhibitory, which predisposed cells to lysozyme and proteinase K digestion.…”

Section: Discussionmentioning

confidence: 99%

“…The six strains from the University of Tasmania (Australia) sub-Antarctic culture collection (four identified as Rhodococcus spp., 1139, 1159, 1163 and 1168, and two as Williamsia spp., 1135 and 1138) [27][28][29] were cultured in a minimal salts media (MSM) consisting of a basal salts solution (BSS) including 7 g/L K 2 HPO 4 , 2 g/L KH 2 PO 4 , 0.25 g/L sea salt (Instant Ocean, Blacksburg, VA, USA), 2 g/L nitrogen source, 10 g/L carbon source, 0.01 g/L FeSO 4 and 10 mg/L thiamine hydrochloride (Sigma-Aldrich, Castle Hill, NSW, Australia). For routine culture of bacterial strains and the control strain R. corynebacteriodes, the nitrogen source used was NH 4 H 2 PO 4 while the carbon source was D-fructose (MSM-F).…”

Section: Bacterial Strains and Culture Conditionsmentioning

confidence: 99%

“…1139, 1159, 1163 and 1168, respectively. Raw data from Macrogen Inc was assembled using ABySS software [27][28][29] and submitted to DDBJ/EMBL/GenBank under the accession numbers: MJEI00000000; MJEJ00000000; LWHK00000000; MJVD00000000; MKKX00000000; and MKKY00000000.…”

Section: Quantity and Purity Of Extracted Dnamentioning

confidence: 99%

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Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species

et al. 2021

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Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii.

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Section: Discussionmentioning

confidence: 99%

Section: Bacterial Strains and Culture Conditionsmentioning

confidence: 99%

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Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species

et al. 2021

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“…Six Corynebacteriales strains originally isolated from Macquarie Island and maintained in the Australian Collection of Antarctic Microorganisms (ACAM) were previously identified by whole-genome sequencing [57][58][59]. These included two novel Williamsia sp.…”

Section: Bacterial Strains and Mediamentioning

confidence: 99%

Application of Thin-Layer Chromatography-Flame Ionization Detection (TLC-FID) to Total Lipid Quantitation in Mycolic-Acid Synthesizing Rhodococcus and Williamsia Species

Nahar

Baker

Nichols

et al. 2020

IJMS

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In addition to cell membrane phospholipids, Actinobacteria in the order Corynebacteriales possess a waxy cell envelope containing mycolic acids (MA). In optimized culture condition, some species can also accumulate high concentrations of intracellular triacylglycerols (TAG), which are a potential source of biodiesel. Bacterial lipid classes and composition alter in response to environmental stresses, including nutrient availability, thus understanding carbon flow into different lipid classes is important when optimizing TAG synthesis. Quantitative and qualitative analysis of lipid classes normally requires combinations of different extraction, derivatization, chromatographic and detection methods. In this study, a single-step thin-layer chromatography-flame ionization detection (TLC-FID) technique was applied to quantify lipid classes in six sub-Antarctic Corynebacteriales strains identified as Rhodococcus and Williamsia species. A hexane:diethyl-ether:acetic acid solvent system separated the total cellular lipids extracted from cells lysed by bead beating, which released more bound and unbound MA than sonication. Typical profiles included a major broad non-polar lipid peak, TAG and phospholipids, although trehalose dimycolates, when present, co-eluted with phospholipids. Ultra-performance liquid chromatography-tandem mass-spectrometry and nuclear magnetic resonance spectroscopy detected MA signatures in the non-polar lipid peak and indicated that these lipids were likely bound, at least in part, to sugars from cell wall arabinogalactan. Waxy esters were not detected. The single-solvent TLC-FID procedure provides a useful platform for the quantitation and preliminary screening of cellular lipid classes when testing the impacts of growth conditions on TAG synthesis.

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Draft Genome Sequences of Two Novel Sub-Antarctic Williamsia Species

Cited by 2 publications

References 9 publications

Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species

Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species

Application of Thin-Layer Chromatography-Flame Ionization Detection (TLC-FID) to Total Lipid Quantitation in Mycolic-Acid Synthesizing Rhodococcus and Williamsia Species

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