The main binding obtained to β-xylans and arabinoxylans is exemplified by families 6 and 22
CBMs from both bacteria (Figure 3.8A). Remarkably C. thermocellum showed a higher number of family 6 CBMs, which exhibited binding to β-xylans and arabinoxylans, whereas R. flavefaciens showed a higher number of family 22 CBMs highly specific to β-xylans and arabinoxylans. In overall, the results reflect the reported specificity for members of family 6 to non-reducing termini of xylo-oligosaccharides, xylans and arabinoxylans38,170–172, as well as the preferential binding of
family 22 CBMs to β-xylans and xylo-oligosaccharides136.
Comparing the oligosaccharide binding patterns, family 22 CBMs exhibited a binding specificity restricted to linear β1,4- or β1,3-β1,4-xylose sequences, with a chain-length dependency from DP-5 up to DP-13, displaying a mono-specific carbohydrate recognition in both bacteria. Additionally, CtCBM22Cthe_1838 showed binding to β1,4-xylose tetrasaccharides with a single
internal α1,2- or α1,3-arabinose branching (Figure 3.8B, probes 168-169). CBMs from family 6 however, presented distinct binding patterns between the two bacteria. C. thermocellum family 6 CBMs showed similar binding specificities to β1,4- or β1,3-β1,4-xylose sequences in the range of DP-3 to DP-13, also recognising β1,4-xylose oligosaccharides with α-arabinose substitutions.
R. flavefaciens RfCBM63747 bound predominantly to arabinoxylan-derived oligosaccharides with
α1,2-arabinose substitutions in the non-reducing terminal xylose (probes 166 and 167).
The binding patterns of family 6 CBMs to the arabinoxylan-derived oligosaccharides evidenced the importance of the free non-reducing β1,4-xylose terminal for recognition, but also the influence of the α-arabinose branching. Although the majority of the CBMs bound only sequences exhibiting the free non-reducing xylose terminal, CtCBM6Cthe_2195 and CtCBM6Cthe_2197 were able to
accommodate sequences with α-arabinose branches in the non-reducing terminal xylose (Figure 3.8A, probes 165 to 167). When comparing the binding intensities, it becomes evident that these two CBMs were able to bind sequences exhibiting α1,2-arabinose substitutions in the non-reducing terminal xylose, whereas the α1,3 configuration was disfavoured. The same binding trend could be observed for probes 168 and 169.
Interestingly, out of the three family 6 CBMs expressed by R. flavefaciens, two are found in large modular proteins associated with family 22 CBMs, RfCBM62649 which did not bind in the
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Figure 3.8. Comparison of carbohydrate-binding specificities of families 6 and 22 CBMs from C. thermocellum and R. flavefaciens FD-1 to xylan sequences. (A) The binding signals of representative
CBMs from each bacterium are depicted as means of fluorescence intensities of duplicate spots at 5 fmol of oligosaccharide probe arrayed (with error bars) and are representative of at least two independent experiments (correspondending to the binding patterns shown in Figure 3.7). Numerical scores are given in
Tables S3.7 and S3.8. The different carbohydrate groups are indicated in the coloured panels. (B) The
sequences of the branched β1,4-xylan(α1,2-arabinose) probes are depicted by microarrays position. Carbohydrate sequence information on these probes is in Chapter 2, Table S2.1.
catalytic modules, which are reported to have α-arabinofuranosidase, β-xylosidase, α-arabinanase and β-galactosidase activity in the degradation of hemicelluloses and pectins, and are frequently found in association with family 6 CBMs173. Given the poly-specificity of CBMs from
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distinct binding specificities than those from C. thermocellum, and for which the target sequences were not included in the microarrays.
In the context of R. flavefaciens cellulosome, the small number of family 6 CBMs and its association with family 22 CBMs, of distinct binding specificities, might point to a complementary function of these modules, evidencing a crucial role of family 6 CBMs in R. flavefaciens.
3.2.5 CBM families for which carbohydrate binding was not identified in the
microarray analyses
C. thermocellum CBMs from families 34, 44, 48 and 54 were not successfully expressed using
the high-throughput strategy and were not analysed in the screening microarrays. Of those CBMs that were expressed and analysed, none of the C. thermocellum CBMs from family 16 or
R. flavefaciens CBMs from families 3, 48 or 63 showed binding in the microarrays (Table S3.2).
The family 16 CBMs from Caldanaerobius polysaccharolyticus (formerly Thermoanaerobacterium
polysaccharolyticum) were reported to bind both β1,4-linked glucose and β1,4-linked mannose
sequences, suggesting the linear β1,4-glucomannan as natural substrate174. The C. thermocellum
CBMs from this family may share similar carbohydrate-binding specificity, explaining why no binding was observed, as glucomannan polysaccharides or oligosaccharides were not included in the microarrays. Although binding was observed with 4 out of the 19 C. thermocellum family 3 CBMs to soluble glucans with a β1,4-linked backbone, these CBMs are characterized to bind to crystalline cellulose with higher affinity160–162. The analysed RfCBM3929,which is associated to a
GH9 cellulase, could be involved in the recognition of insoluble β1,4-glucans154. Family 48 CBMs
have reported activities towards starch and glycogen, binding to linear and cyclic sequences of α1,4- and α1,6-linked glucose175. As only linear α-glucans were included in the microarrays, might
be possible that the RfCBM48s require branched or cyclic sequences for binding recognition. The
Bacillus subtilis CBM63 is reported to be associated with expansin module EXLX1 and to mediate
the expansin binding to cell wall cellulose176. The analysed RfCBM632821, being also linked to an
expansin module, may have similar activity towards different forms of cellulose.
3.2.6 CBMs spectrum of carbohydrate recognition reflects the bacteria’s
ecological niche
C. thermocellum CBMs showed broader binding patterns, while having a larger repertoire of CAZy
families that include a higher number of CBMs specific for recognition of β-glucans. This larger cohort of C. thermocellum CBMs, not only from different CAZy families but within the same family (such as the high numbers of family 3 and 6 CBMs), may contribute for its high efficiency in degradation of a wide range of plant cell wall polysaccharides. In addition, the elevated number of family 50 CBMs (LysMs) may confer to C. thermocellum an advantage for survival in the extreme conditions of the ecological niche it resides,as LysMs have also been reported to play a role in the development of spores in other spore-forming bacteria, such as Bacillus subtilis42,177.
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R. flavefaciens exhibited a more restricted carbohydrate binding recognition and, although
apparently expressing less CAZy families, holds a greater number of CBMs targeting hemicelluloses β-xylans, β-mannans and pectic α-arabinans and galactans. Noteworthy, is the evidence that R. flavefaciens cellulosome contains a large number protein modules of unknown sequence homology to assigned CAZy families, six of which were in the course of this thesis reported by Venditto and colleagues59 to exhibit carbohydrate binding and were assigned to the
new CBM families 75 to 80. These CBMs target as major substrates the hemicelluloses xyloglucan and β-mannans, β1,4- and mixed-linked β1,3-1,4-glucans, and pectins. In this context, the data reported here complements this study in that the complex R. flavefaciens cellulosome seems to incorporate an extended CBM repertoire that promotes the efficient plant cell wall hemicellulose and pectin degradation, even though expressing a small number of CBMs that specifically target crystalline cellulose. Considering its highly dynamic and populated ecological niche, R. flavefaciens may also benefit from a cooperative relationship with other members of the mammalian rumen microbiome, such as Ruminococcus albus and Fibrobacter succinogenes responsible for the breakdown of the recalcitrant structure of cellulose178, ensuring its substrate
acquisition and survival.
3.3 Conclusions
In the present work, different patterns of polysaccharide and oligosaccharide binding by
C. thermocellum and R. flavefaciens CBMs were revealed and novel specificities were assigned.
Although C. thermocellum CBMs have been more extensively studied and characterized, new CBM carbohydrate binding specificities were identified for CBMs families 25, 42 and 50. For
R. flavefaciens, ligand-specificities were obtained for 21 CBMs from families 4, 6, 13, 22 and 35.
Aiming to decipher the complete CBMome of R. flavefaciens, analysis of the remaining CBM families in the oligosaccharide microarrays described in this Chapter and Chapter 2, is required. Overall, the combined use of high-throughput methodologies allowed to explore the function of
C. thermocellum and R. flavefaciens CBMomes, revealing that the two bacteria present CBMs
expressing different carbohydrate-binding specificities, which reflect at some extent the different polysaccharides that each bacterium may encounter in its ecological niche. This comparative study of two bacteria residing in different ecological niches, provides experimental evidence supporting that substrate availability in different habitats may modulate the evolutionary selection of CAZymes to present modules with distinct carbohydrate ligand specificities.
This study also highlights the importance of developing high-throughput methodologies to study these complex systems and unravel carbohydrate recognition. The approach of using in parallel polysaccharide and oligosaccharide microarrays, allows detailed characterization of the specificities of CBMs. While polysaccharide microarrays enable carbohydrate-binding to be assigned, oligosaccharide microarrays can reveal subtle differences in binding profiles and chain-length dependencies, which enables to differentiate between the different topologies of
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CBMs binding sites and their functional types. The information obtained from the carbohydrate microarray analyses is crucial to assess the structural characterization of the interactions of CBMs with their oligosaccharide ligands. These integrative studies will be important to elucidate cellulolytic capabilities of these bacteria at the molecular level. To this end, up to 13 CBMs belonging to different CAZy families of both bacteria were selected for large-scale protein expression and purification. Preliminary conditions were obtained for CBMs of families 25 and 50 from C. thermocellum and families 6, 13, and 62 from R. flavefaciens. Structural characterization of the carbohydrate-binding specificity of CBMs from C. thermocellum family 11 and 50 and
R. flavefaciens family 13 will be explored in Chapters 4, 5 and 6, respectively.
3.4 Experimental procedures
3.4.1 Monoclonal antibodies, CBMs and lectins used for microarray quality
control
Details on the plant cell wall carbohydrate-directed monoclonal antibodies, CBMs with characterised carbohydrate-binding specificities and plant lectins used for microarray quality control are given in Table S2.3 and section 2.5.1 in Chapter 2.