Taxono-genomics description of Olsenella lakotia SW165 T sp. nov., a new anaerobic bacterium isolated from cecum of feral chicken

Abstract

Background: The microbial community residing in the animal gastrointestinal tract play a crucial role in host health. Because of the high complexity of gut microbes, many microbes remain unclassified. Deciphering the role of each bacteria in health and diseases is only possible after its culture, identification, and characterization. During the culturomics study of feral chicken cecal sample, we cultured a possible novel strain SW165 ^T. Methods: For the possible novel strain SW165 ^T, phenotypic characterization was performed using colony morphology, Gram staining, growth in different temperature and pH and motility. Biochemical assays included carbon source utilization, enzymatic activity, cellular fatty acids and short chain fatty acid production. 16S rRNA sequencing and whole genome sequencing and comparison was performed for genetic analysis. Results: This strain was isolated from cecal content of feral chickens in Brookings, South Dakota, USA. Phylogenetic analyses based on 16S rRNA gene sequence revealed that the closest valid neighbor was Olsenella profusa DSM 13989 ^T (96.33% similarity) within the family Atopobiaceae. Cells were Gram-strain-positive and obligately anaerobic bacilli in chains. The optimum temperature and pH for the growth of the microorganism were 37-45 ^oC and pH 6.0-7.5 respectively. This strain produced acetic acid as the primary fermentation product. Major fatty acids were C _12:0, C _14:0, C _14:0 DMA and summed feature 1 (C _13:1 at 12-13 and C _14:0 aldehyde). Strain SW165 ^T exhibited a genome size of 2.43 Mbp with a G+C content of 67.59 mol%, which is the second highest G+C content among members of the genus Olsenella. The digital DNA-DNA hybridization and OrthoANI values between SW165 ^T and DSM 13989 ^T were only 17.6 ± 5.3 and 74.35%, respectively. Conclusion: Based on the phenotypic, biochemical, and genomic analyses, we propose the new species of the genus Olsenella, and name it Olsenella lakotia SW165 ^T sp. nov., (=DSM 107283 =CCOS 1887) as the type strain.

Keywords: Olsenella lakotia SW165T sp. nov.; chicken gut microbiota; culturomics; taxono-genomics.

Figure 1.. 16S rRNA based neighbor-joining tree of SW165 ^T and its neighbors.

Tree shows phylogenetic position of Olsenella lakotia DSM 107283 ^T and closely related species in the family Atopobiaceae. GenBank accession numbers of the 16S rRNA gene sequences are given in parentheses. Black circles indicate that the corresponding branches were also recovered both by maximum-likelihood and maximum parsimony methods. Bootstrap values (based on 1000 replications) greater than or equal to 70% are shown in percentages at each node. Bar, 0.01 substitutions per nucleotide position.

Figure 2.. Scanning electron micrograph of O. lakotia SW165 ^T.

Cells were anaerobically cultured for 24 hours at 37 ^o C in BHI-M medium. Bar, 2 μm; uncropped/unedited image.

Figure 3.. Graphical circular map of O. lakotia SW165 ^T genome.

From outside to the center: coding sequences on the forward strand (CDS +), coding sequences on the reverse strand (CDS -), tRNAs, rRNAs, GC content, and GD skew.

Figure 4.. Average nucleotide Identity comparison of O. lakotia SW165 ^T and closely related strains.

Heatmap represents OrthoANI values generated using OAT software between O. lakotia and related taxa with valid taxonomy.

Figure 5.. Distribution of functional features of predicted coding sequences of O. lakotia SW165 ^T and its neighbors.

The functional features were predicted based on the clusters of orthologous groups. Heatmap was generated from genome annotation of individual species by RAST using Explicet software.