UC Riverside

Microbial populations observed in the microbiome of Acrobeloides maximus consistently comprised a set of different microorganisms compared to the microbiome of Caenorhabditis elegans or the metagenome of the soil sample to which nematodes were exposed. Using 16S rRNA gene amplification, the bacterial genera Ochrobactrum, Pedobacter, and Chitinophaga were identified at significant levels within the A. maximus communities only and were found to be consistently present during each of the three sampling periods over six months. We were able to isolate and grow colonies of Ochrobactrum and Pedobacter from the microbiome of the nematode on Lysogeny Broth-Miller (LB) and Yeast Extract (YE) agar plates. We were unable to isolate the associate from the genus Chitinophaga. The results obtained suggest a possible specific and persistent association between A. maximus and the two bacterial isolates we were able to culture.

With fluorescence in situ hybridization (FISH) with probes specific for Ochrobactrum and Pedobacter, we were able to stain these bacterial cells and show that they reside within the intestinal tract of A. maximus. It is possible that these gut bacteria may be mutualistic by providing A. maximus with essential metabolites, by aiding the nematode in the digestion of other bacteria, or by providing it with protection against pathogens.

Using 16S rRNA amplification and sequencing, three bacterial strains belonging to the genera Ochrobactrum, Pedobacter, and Chitinophaga were detected at significant levels within cultured Acrobeloides maximus populations exposed to soil for 24 hours before extraction and re-inoculation on agar. All three strains were found to persist in re-inoculated A. maximus cultures over a period of six months. For each strain, sequences from our A. maximus associated clones along with relevant 16S rRNA sequences from congeners or related genera available in GenBank were subjected to comprehensive phylogenetic analyses. Sequences were aligned using the multiple sequence alignment algorithms implemented in T-Coffee, MAFFT, Kalign, and WebPRANK, and phylogenetic trees were constructed using Bayesian and maximum-likelihood methods. Similarities and differences among the different alignment and tree-building methods suggest tree building methods with T-Coffee performed more poorly while maximum-likelihood methods with MAFFT and Kalign produced more mutually consistent topologies that correspond better to recognized genus boundaries. Inferences are made about the known species that are phylogenetically closest to each of our three strains. Our Ochrobactrum strains appear to be most closely related to Ochrobactrum tritici, while our Chitinophaga strains are most similar to a bacterium previously identified as “Flexibacter cf. sancti.” Our Pedobacter strains seem to be a previously undescribed sphingobacterium within the genus.