Phylogenetic analysis of 16S rRNA gene sequences
The taxonomy of the isolated strains was determined based on their 16S rRNA gene sequences after an initial taxonomic analysis of their whole genome sequences.
First, the genomes of KH365_2T and KH569_7 were uploaded to the Type Strain Genome Server (TYGS) (Meier-Kolthoff and Göker 2019) to check whether the strains are classified as members of an already known bacterial species. The genome sequences of KH365_2T and KH569_7 did not match any sequence in the database and were identified as members of a potentially novel species of the genus Bacteroides. These results were further supported by pairwise digital DNA–DNA hybridization (dDDH) values between the two strains and their 19 most closely related Bacteroides type strains (automatically determined by TYGS), which ranged from 21.7 to 49.2% and were all below the threshold of microbial species delineation (70%). The pairwise dDDH value between the strains KH365_2T and KH569_7 was 87% (Table S1), indicating that they belonged to the same species (Meier-Kolthoff et al. 2014).
To build a phylogeny of Bacteroides species including the two strains KH365_2T and KH569_7, 16S rRNA gene sequences were extracted and compared against those of 35 Bacteroides type strains with validly published names (www.bacterio.net/genus/bacteroides.html). KH365_2T and KH569_7 clustered in a neighbor-joining tree within the genus Bacteroides (cluster represented by KH365_2T) (Fig. 1). Furthermore, they formed a clade with Bacteroides uniformis ATCC 8492 T and Bacteroides rodentium JCM 16496 T. These results were consistent with the topologies of maximum-likelihood and maximum-parsimony trees (Figs. S1 and S2). Pairwise sequence analysis of the full-length 16S rRNA gene sequences between the novel strains and other Bacteroides type strains revealed that KH365_2T and KH569_7 had the highest sequence similarity to B. uniformis ATCC 8492 T (accession no. AB050110, 96.83%), followed by B. rodentium JCM 16496T (AB547646, 96.75%) and Bacteroides fluxus YIT 12057T (AB490802, 94.33%). These values were below the species delineation threshold of 98.7% (Goebel and Stackebrandt 1994). Moreover, 16S rRNA gene sequence similarity between strains KH365_2T and KH569_7 was 99.86%, supporting the results of the whole genome analysis by TYGS that these two strains might represent the same novel Bacteroides taxon. A comparison between the herein described strains KH365_2T and KH569_7, and the strain Bacteroides sp. NM69_E16B revealed a sequence identity above the species delineation threshold (99.8%), indicating that all three strains belong to the same novel taxon.
To study genomic features of KH365_2T and KH569_7, whole genomes were sequenced and analyzed. The genomic differences between the two strains to other Bacteroides species, which had already been observed in 16S rRNA gene sequences (Fig. 1), were additionally determined at the level of the whole genome. The dDDH values between KH365_2T and KH569_7 and their 19 most closely related Bacteroides type strains ranged from 21.7 to 49.2%, which is below the threshold of microbial species delineation (70%) (Meier-Kolthoff et al. 2014). The pairwise dDDH value between the two novel strains was 87% (Table S1) and thereby above the threshold. Similarly, OrthoANIu values between the novel strains and type strains of closely related species were below the species delineation thresholds, ranging from 80.07 to 80.22% similarity with B. fluxus YIT 12057T and 92.01% to 91.85% similarity with B. uniformis ATCC 8492T (Table S1). These results further support the assignment of KH365_2T and KH569_7 to the same novel species. Moreover, the comparison of dDDH and orthoANIu confirm the classification of Bacteroides sp. NM69_E16B in the same species as the herein described strains (Table S2).
The draft genomes of KH365_2T and KH569_7 were 4 164 660 bp and 4 177 920 bp long, with 46.02% and 46.03% mol G + C content, respectively (Table S1). Coding DNA sequences (CDSs), rRNAs, and tRNAs were annotated in the draft genomes (Table S3). A total of 3886 and 3903 CDSs were predicted for KH365_2T and KH569_7, respectively. Further genome mining revealed 101 proteins involved in polysaccharide transport and degradation encoded in the genome of KH365_2T, while only 75 were identified in the genome of KH569_7. Moreover, several beta-lactamases were detected in the draft genomes of both strains, potentially indicating resistance to some antibiotics. The numbers of identified rRNAs and tRNAs in KH365_2T were 6 and 66, respectively. Strain KH569_7 had 5 rRNAs and 48 tRNAs in its genome. These results are within the range of the number of rRNAs and tRNAs identified in closely related Bacteroides species (Table S3). Both strains, KH365_2T and KH569_7, harbored two predicted gene clusters each for the synthesis of secondary metabolites: ribosomally synthesized and posttranslationally modified peptide classes (RiPPs) and respective recognition elements.
Taken together, whole genome sequences confirm the classification of the two strains distinct from known Bacteroides species, predict a function in polysaccharide metabolism among others, and reveal genomic differences between the two strains.
To characterize the morphology of KH365_2T and KH569_7, we imaged bacterial colonies and cells microscopically. The colonies of KH365_2T were smooth, creamy, circular, convex, and approximately 0.8–1.0 mm in diameter (Fig. 2a). Their appearance was similar to colonies of B. uniformis ATCC 8492T (Fig. 2a). The colony morphology of KH569_7 was analogous to the strain KH365_2T and B. uniformis type strain, except for a smaller colony size (0.6–0.8 mm in diameter) and slightly brighter color when grown under the same conditions (Fig. 2a). The difference in colony size might be explained by differences in growth, which are also observed when growing the strains in liquid media (Fig. 2b).
Next, TEM was performed to analyze the cell morphology and ultrastructure (Figs. 2c, d; S3, S4). Negative staining of live bacteria that had been cultured for 24 h revealed rod-shaped cells in all three strains in the size range of 1.62–3.38 × 0.80–0.85 µm for the strain KH365_2T, 1.00–2.60 × 0.80–0.87 µm for the strain KH569_7, and 1.13–2.08 × 0.85–0.92 µm for B. uniformis ATCC 8492 T. Notably, some B. muris KH365_2 cells could grow unusually long, up to 9 µm in length, which was not detected in the other two strains (Fig. S3). We observed dividing cells showing the typical constriction characteristic for the division of Gram-negative bacteria (Fig. 2c). All three strains produced outer membrane vesicles (Fig. 2c). Thin sections of resin embedded bacteria were used to characterize the ultrastructure of the cell envelope (Figs. 2d; S4). In all three strains (KH365_2T, KH569_7 and B. uniformis ATCC 8492T), the cell envelope consists of multiple layers: the cytoplasmic membrane, the periplasm, the outer membrane, and the microcapsule external to the outer membrane. Between independent samples of the same strain, and even within single populations, we observed bacteria with different structures of the microcapsule (Fig. 2d). This suggests that the phase variation in the capsule production observed earlier in intestinal Bacteroides species (Coyne and Comstock 2008) is also present in the newly described strains. The capsule could appear as i) one fringed layer (Fig. 2d i), ii) an electron dense layer with an additional, peripheral compact layer (AL, based on a description by Kornman and Holt (1981)) (Fig. 2d ii), or iii) like in (ii), but with an extra fringed layer beyond the AL (Fig. 2d iii). In KH365_2T and B. uniformis ATCC 8492T, all three variations of the capsule were observed, whereas in KH569_7, only (ii) and (iii) were detected (Fig. S4).
In brief, these images show rod-shaped cells, illustrate the ultrastructure of the multi-layered cell envelope, reveal the production of outer membrane vesicles, and demonstrate variable production of the microcapsule in a single bacterial population. The two B. muris strains share most morphological features in common and yet differ slightly.
Next, we studied the physiological and biochemical characteristics of the two strains. First, their Gram-stain was assessed as a basic parameter to differentiate between bacteria. KH365_2T and KH569_7 were Gram-negative (Table 1), which was to be expected based on the ultrastructure of the cell envelope (Fig. 2d) and is consistent with Gram-negative staining of B. uniformis ATCC 8492T and other members of the genus Bacteroides.
Second, multiple measurements were performed to study the conditions under which the strains grow. When grown as stab cultures in aerobic conditions, KH365_2T, KH569_7, and B. uniformis ATCC 8492T strains grew only at the bottom of the tube, suggesting that these bacteria are strictly anaerobic. The two strains KH365_2T and KH569_7 were calatase- and oxidase-negative, as was the closely related B. uniformis ATCC 8492T strain (Table 1). The KH365_2T strain was able to grow in the presence of 0.5–2.0% (w/v) NaCl, similar to B. uniformis ATCC 8492T (optimal growth at 0.5–1% (w/v)), whereas the KH569_7 strain could tolerate as little as 0.5% (w/v) NaCl (Table 1). Similar to B. uniformis ATCC 8492T, optimal growth of KH365_2T and KH569_7 was observed at 32–37 °C; while KH365_2T was able to grow at 20–42 °C and KH569_7 at 20–37 °C (Table 1). All three strains grew at pH 5–10 with slight differences in the optimum pH (Table 1).
Third, the metabolic repertoire of KH365_2T and KH569_7 was systematically studied by performing a series of Biolog assays. We measured the extent to which 95 carbon sources were utilized by the novel strains and B. uniformis ATCC 8492T (Fig. S5). Two metabolites, L-alanyl-l-histidine and i-erythritol, were differently utilized by KH365_2T and KH569_7, compared to their closest relative B. uniformis ATCC 8492T, i.e., these are metabolites that are better utilized by B. uniformis ATCC 8492T than by either of the novel strains (Fig. 2e). We further observed differences in the metabolism between the two strains of the novel species. Nineteen different metabolites, including lactic acid and some sugars, among others, were differentially utilized by KH365_2T and KH569_7 (Fig. 2f), indicating a high degree of intra-specific diversity. Notably, all 19 of these metabolites were significantly better utilized by KH365_2T than by KH569_7, and 12 of them were utilized similarly by KH365_2T and B. uniformis ATCC 8492T.
Finally, we checked for motility of the strains. All were non-motile (Table 1), consistent with reports on other Bacteroides species (Lakhdari et al. 2011).
In summary, the physiology of the two strains is largely consistent with what might be expected from a Bacteroides species. Both strains lack some metabolic features of the closely related B. uniformis and show considerable intraspecific variation.