Sporulation capability and amylosome conservation among diverse human colonic and rumen isolates of the keystone starch-degrader Ruminococcus bromii

Abstract

Ruminococcus bromii is a dominant member of the human colonic microbiota that plays a 'keystone' role in degrading dietary resistant starch. Recent evidence from one strain has uncovered a unique cell surface 'amylosome' complex that organizes starch-degrading enzymes. New genome analysis presented here reveals further features of this complex and shows remarkable conservation of amylosome components between human colonic strains from three different continents and a R. bromii strain from the rumen of Australian cattle. These R. bromii strains encode a narrow spectrum of carbohydrate active enzymes (CAZymes) that reflect extreme specialization in starch utilization. Starch hydrolysis products are taken up mainly as oligosaccharides, with only one strain able to grow on glucose. The human strains, but not the rumen strain, also possess transporters that allow growth on galactose and fructose. R. bromii strains possess a full complement of sporulation and spore germination genes and we demonstrate the ability to form spores that survive exposure to air. Spore formation is likely to be a critical factor in the ecology of this nutritionally highly specialized bacterium, which was previously regarded as 'non-sporing', helping to explain its widespread occurrence in the gut microbiota through the ability to transmit between hosts.

Figure 1

Comparison of five R. bromii genomes. A. Pan‐genome analysis of core, accessory and unique genes among five R. bromii strains. A total of 735 core orthologous groups (OGs), 1456 variable OGs and 2240 unique genes were detected in the R. bromii pan‐genome. Overall, the R. bromii YE282 strain genome has the highest number of unique genes (1561) compared to the human R. bromii genomes suggesting that these may be associated with colonization of the rumen. B. Genomic comparison of four R. bromii genomes to reference strain ATCC27255. Diagram represents BLASTn results of each genome against ATCC2755 strain with results rendered using the BRIG program. The inner circle represents the reference genome R. bromii ATCC27255. Each genome is colour coded as indicated by the legend. Relative shading density (from darker to lighter) within each circle represents relative levels of nucleotide homology. White regions indicate regions with no identity to the reference. The location of genes encoding GH13 enzymes (Amy1–17) is also indicated. C. Representation (number per genome) of glycoside hydrolase families encoded by R. bromii genomes.

Figure 2

Phylogenetic tree based on the amino acid sequences of GH13 catalytic domains of the 9 extracellular GH13 enzymes (that carry signal peptides) from 5 R. bromii strains. The sequences of the GH13 genes shown here from the five R. bromii strains fall into 9 significant clusters, with sequences from the four human R. bromii strains clustering more closely with each other than the YE282 rumen strain. YE282 sequences are identified here by their numerical identifiers in the JGI database, whereas genes from the human strains are prefixed by the strain designation. Boxes indicate GH13 domains that are associated with dockerins. Bootstrap values, expressed as a percentage of 1000 replications, are given at the branching nodes. This tree is unrooted and reconstructed using the maximum‐likelihood method. The scale bar refers to the number of amino acid differences per position. Similar clustering was also observed for the remaining GH13 enzymes listed in Table 1 that are common to all five strains (not shown).

Figure 3

Cohesin‐dockerin binding measured by ELISA. A. A microtiter plate was coated with XyndocAmy16. Positive interactions of the Amy16 dockerin were observed with coh1 and 3; coh1, 2, 3 and 4 are from Sca1 (Amy4), 2, 3 and 4, respectively. B. ELISA plates were coated with CBM‐Coh from Sca5. Positive interactions of the cohesin from Sca5 were observed with Xyndocs 14720, 16032, Amy4, Amy12 and Amy9, low interaction was also observed with Xyndoc Amy10. (Please note that to allow comparison the CBL numbering for Xyndocs given here is consistent with that given previously (Ze et al., 2015) and does not refer to the L2–63 genome re‐annotation). Error bars indicate SD from the mean of duplicate samples from one experiment. C. Recombinant gfp‐Amy4doc protein incubated with EDTA‐pretreated, washed R. bromii L2–63 cells in the presence of Ca²⁺. (D) as (C) but in the absence of Ca²⁺. E. R. bromii L2–63 cells in the absence of gfp‐amy4doc.

Figure 4

Updated model for cell‐bound and cell‐free amylosome complexes in R. bromii L2–63. The Amy4 and Amy9 enzymes are likely to bind to the cell surface via the Sca2 scaffoldin protein. Amy4 has then the potential to self‐aggregate through interactions between its own cohesin and dockerin or to integrate Amy9, Amy10 or Amy16. Further complexes are likely to form between the Amy4, Amy9, Amy10, Amy12, Amy16 and either the Sca3 or Sca4 proteins to form cell‐free amylosomes. In addition, Amy4, Amy9, Amy10, Amy12 can be integrated to the first cohesin of Sca5 and attached to the cell‐surface. The binding specificities of cohesin modules of the second Sca5 cohesin (shown in light grey) have yet to be determined.

Figure 5

Carbohydrate utilization in R. bromii strains. Total sugar utilized and the concentration of free soluble reducing sugar (glucose equivalents) are shown after 48 h incubation in M2 medium containing (A) RS2 (High‐maize 958) or (B) RS3 starch (Novelose 330). Data plotted in graphs are the mean ± SD OD readings (OD₄₉₀ for total sugar assay and OD₄₁₅ for reducing sugar assay) of three biological replicates and three technical replicates for each time‐point studied for each strain. (C) Organization of the galactose operon in the human R. bromii strains (P indicates likely promoters and the hairpin indicates the likely transcriptional terminator). (D) Growth of the five R. bromii strains with soluble potato starch, maltose, fructose, galactose, lactose, glucose and arabinose (0.2% w/v) as sole sources of carbon. Data plotted in graphs are the mean OD₆₅₀ readings of three replicates of each strain grown on different sugar substrates. As the SD values were very small in all cases they were not plotted on to the graph.

Figure 6

Sporulation gene signature and TEM of R. bromii sporulation. (A) A set of 65 genes with known sporulation function were detected in the genome of R. bromii L2–63 strain. These genes corresponded to: initiation of spore formation (Stage I), asymmetrical division into a larger mother cell and a smaller forespore (Stage II), engulfment of the forespore by the mother cell (Stage III), formation of the spore cortex (Stage IV), deposition of the spore coat (Stage V) followed by lysis of the mother cell and release of the endospore (Stage VI). A set of 8 genes related to germination of the endospore were also detected. Spo0A, which is a critical transcription factor to initiate sporulation and the specialized sporulation sigma factors (σH, σF, σE, σG and σK; small proteins that direct RNA polymerase to specific sites on DNA to initiate gene expression) along with the regulators of these sigma factors (such as spoIIAA, spoIIGA and the spoIIIA operon) were also present. Key regulatory proteins involved in chromosome partitioning, belonging to the soj (parA_1, parA_2, parA_3,) and spo0J (parB_1, parB_2, parB_3, parB_4, parB_5) families were also detected. In the absence of spoJ, soj is known to negatively regulate expression of several sporulation genes by binding to the promoter regions and inhibiting transcription indicating a tightly regulated energy‐intensive process for survival. Comparison with core sporulation and germination related genes from Clostridium difficile strain 630 and Bacillus subtilis strain 168 described recently by Browne and colleagues (2016) showed that all the key sporulation genes were present. (B) TEM image of a R. bromii L2–63 strain after 72 h growth on M2S medium. (C and D) Endospores were visible from R. bromii cells (E) Release of endospores from R. bromii after lysis of the mother cell. (F) Endospores released into the surrounding medium.