Host-Gut Microbiome Metabolic Interactions in PFAS-Impacted Freshwater Turtles (Emydura macquarii macquarii).

Per-and polyfluoroalkyl substances (PFAS) are a growing concern for humans, wildlife, and more broadly, ecosystem health. Previously, we characterised the microbial and biochemical impact of elevated PFAS on the gut microbiome of freshwater turtles (Emydura macquarii macquarii) within a contaminated catchment in Queensland, Australia. However, the understanding of PFAS impacts on this species and other aquatic organisms is still very limited, especially at the host-gut microbiome molecular interaction level. To this end, the present study aimed to apply these leading-edge omics technologies within an integrated framework that provides biological insight into the host turtle-turtle gut microbiome interactions of PFAS-impacted wild-caught freshwater turtles. For this purpose, faecal samples from PFAS-impacted turtles (n = 5) and suitable PFAS-free reference turtles (n = 5) were collected and analysed. Data from 16S rRNA gene amplicon sequencing and metabolomic profiling of the turtle faeces were integrated using MetOrigin to assign host, microbiome, and co-metabolism activities. Significant variation in microbial composition was observed between the two turtle groups. The PFAS-impacted turtles showed a higher relative abundance of Firmicutes and a lower relative abundance of Bacteroidota than the reference turtles. The faecal metabolome showed several metabolites and pathways significantly affected by PFAS exposure. Turtles exposed to PFAS displayed altered amino acid and butanoate metabolisms, as well as altered purine and pyrimidine metabolism. It is predicted from this study that PFAS-impacted both the metabolism of the host turtle and its gut microbiota which in turn has the potential to influence the host's physiology and health.

Perturbation of the gut microbiome in wild-caught freshwater turtles (Emydura macquarii macquarii) exposed to elevated PFAS levels.

Per- and polyfluoroalkyl substances (PFAS) are environmentally persistent and pervasive. Understanding the toxicity of PFAS to wildlife is difficult, both due to the complexity of biotic and abiotic perturbations in the taxa under study and the practical and ethical problems associated with studying the impacts of environmental pollutants on free living wildlife. One avenue of inquiry into the effects of environmental pollutants, such as PFAS, is assessing the impact on the host gut microbiome. Here we show the microbial composition and biochemical functional outputs from the gut microbiome of sampled faeces from euthanised and necropsied wild-caught freshwater turtles (Emydura macquarii macquarii) exposed to elevated PFAS levels. The microbial community composition was profiled by 16S rRNA gene sequencing using a Nanopore MinION and the biochemical functional outputs of the gut microbiome were profiled using a combination of targeted central carbon metabolism metabolomics using liquid chromatography coupled to a triple quadrupole mass spectrometer (LC-QqQ-MS) and untargeted metabolomics using liquid chromatography coupled to a quadrupole time of flight mass spectrometer (LC-QToF-MS). Total PFAS was measured in the turtle serum using standard methods. These preliminary data demonstrated a 60-fold PFAS increase in impacted turtles compared to the sampled aquatic environment. The microbiome community was also impacted in the PFAS exposed turtles, with the ratio of Firmicutes-to-Bacteroidetes rising from 1.4 at the reference site to 5.5 at the PFAS impacted site. This ratio increase is indicative of host stress and dysfunction of the gut microbiome that was correlated with the biochemical metabolic function data, metabolites observed that are indications of stress and inflammation in the gut microbiome. Utilising the gut microbiome of sampled faeces collected from freshwater turtles provides a non-destructive avenue for investigating the impacts of PFAS in native wildlife, and provides an avenue to explore other contaminants in higher-order taxa within the environment.

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

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.

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

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.

Draft Genome Sequences of Two Lactobacillus casei Strains Isolated from Cheddar Cheese and a Fermented Milk Drink.

MiSeq Illumina shotgun sequencing technology was used to sequence two Lactobacillus casei strains, designated strains GCRL 163 and MJA 12. The estimated genome sizes for GCRL 163 and MJA 12 were 2.9 Mb and 3.1 Mb, with 46.35% and 46.31% GC contents, respectively.

Draft Genome Sequences of Two Novel Sub-Antarctic Williamsia Species.

Illumina MiSeq shotgun sequencing technology was used to sequence the genomes of two novel sub-Antarctic Williamsia species, designated strains 1135 and 1138. The estimated genome sizes for strains 1135 and 1138 are 5.99 Mb and 6.08 Mb, respectively. This genome sequence information will aid in understanding the lipid metabolic pathways of cold-tolerant Williamsia species.

Draft Genome Sequences of Three Sub-Antarctic Rhodococcus spp., Including Two Novel Psychrophilic Genomospecies.

The draft genome sequences of three sub-Antarctic Rhodococcus sp. strains-1159, 1163, and 1168-are reported here. The estimated genome sizes were 7.09 Mb with a 62.3% GC content for strain 1159, 4.45 Mb with a 62.3% GC content for strain 1163, and 5.06 Mb with a 62.10% GC content for strain 1168.

Draft Genome Sequence of Subantarctic Rhodococcus sp. Strain 1139.

The draft genome sequence of subantarctic Rhodococcus sp. strain 1139 is reported here. The genome size is 7.04 Mb with high G+C content (62.3%) and it contains a large number of genes involved in lipid synthesis. This lipid synthesis system is characteristic of oleaginous Actinobacteria, which are of interest for biofuel production.