Isolation and sequence-based characterization of a koala symbiont: Lonepinella koalarum

Abstract

Koalas (Phascolarctos cinereus) are highly specialized herbivorous marsupials that feed almost exclusively on Eucalyptus leaves, which are known to contain varying concentrations of many different toxic chemical compounds. The literature suggests that Lonepinella koalarum, a bacterium in the Pasteurellaceae family, can break down some of these toxic chemical compounds. Furthermore, in a previous study, we identified L. koalarum as the most predictive taxon of koala survival during antibiotic treatment. Therefore, we believe that this bacterium may be important for koala health. Here, we isolated a strain of L. koalarum from a healthy koala female and sequenced its genome using a combination of short-read and long-read sequencing. We placed the genome assembly into a phylogenetic tree based on 120 genome markers using the Genome Taxonomy Database (GTDB), which currently does not include any L. koalarum assemblies. Our genome assembly fell in the middle of a group of Haemophilus, Pasteurella and Basfia species. According to average nucleotide identity and a 16S rRNA gene tree, the closest relative of our isolate is L. koalarum strain Y17189. Then, we annotated the gene sequences and compared them to 55 closely related, publicly available genomes. Several genes that are known to be involved in carbohydrate metabolism could exclusively be found in L. koalarum relative to the other taxa in the pangenome, including glycoside hydrolase families GH2, GH31, GH32, GH43 and GH77. Among the predicted genes of L. koalarum were 79 candidates putatively involved in the degradation of plant secondary metabolites. Additionally, several genes coding for amino acid variants were found that had been shown to confer antibiotic resistance in other bacterial species against pulvomycin, beta-lactam antibiotics and the antibiotic efflux pump KpnH. In summary, this genetic characterization allows us to build hypotheses to explore the potentially beneficial role that L. koalarum might play in the koala intestinal microbiome. Characterizing and understanding beneficial symbionts at the whole genome level is important for the development of anti- and probiotic treatments for koalas, a highly threatened species due to habitat loss, wildfires, and high prevalence of Chlamydia infections.

Characterization of the juvenile koala gut microbiome across wild populations

“…It is also possible that there are undescribed tannase genes present in the koala gut microbiome that are dissimilar from those currently described and thus were not identified in our analysis. For instance, in a recent study of L. koalarum genomes, no tannase genes could be identified, despite the in vitro assays suggesting that they should be present (Dahlhausen et al, 2020). Despite the capacity of some microbes to degrade tannin-protein complexes, the nutritional significance of this for many herbivores is uncertain, as the antinutritional functions of dietary tannins persist and influence diet selection and nutritional performance of koalas and other herbivores (DeGabriel et al, 2009).…”

Lonepinella Sp. Isolated From Wound Infections of Koalas

“…Phylogenetic analysis of the draft genome assemblies using the GTDB platform clustered the two koala isolates closely to the L. koalarum type strain as well as to the previously isolated and sequenced isolate UCD_LQP1 obtained from the feces of a female healthy koala at the San Francisco Zoo, San Francisco, California, USA (Dahlhausen et al 2020; Fig. 1, GenBank accession number GCF_008723255.1).…”

“…A phylogenetic tree was inferred using the FastTree version 2.1.10, implemented in the de novo workflow, with the 45,555 GTDB genomes from GTDB-Tk reference data v.r202. This included a total of 133 genomes from the family Pasteurellaceae plus the two assemblies from the current study and the draft genome assembly of L. koalarum strain UCDLQP1 (Dahlhausen et al 2020), with genus Gallibacterium as the outgroup. The average nucleotide identity (ANI) between the genome of the isolates and that from L. koalarum –type strain was calculated using the ani_rep workflow in the GTDB-Tk.…”

Individuality and stability of the koala (Phascolarctos cinereus) faecal microbiota through time

“…L koalarum has been shown to degrade tannin-protein complexes and was first isolated and described by Osawa et al (1995) . This bacterium has received interest due to its positive association with koala survival during antibiotic treatment for Chlamydia ( Dahlhausen et al, 2018 ; Dahlhausen et al, 2020 ). However, these inter-population findings should be treated with caution, as our population-level comparison is confounded by captive (Cleland) vs wild (Mountain Lagoon), which can influence faecal microbiota composition in some vertebrate species ( Blyton et al, 2022b ; Kohl, Skopec & Dearing, 2014 ; Alberdi, Martin Bideguren & Aizpurua, 2021 ).…”