Algunas conclusiones sobre la dinámica y la complejidad de la administración

The genome of M. pectinilyticus was searched against currently available protein databases and the CAZyme database to identify dockerins, cohesins, and scaffoldins which constitute the assembly of cellulosome. Only 2 dockerins were found - in hypothetical proteins encoded by B9O19_388 and B9O19_1577 - and 3 cohesin-like domains - in B9O19_1578 and B9O19_1579 - indicating these domains likely occurred outside the cellulosome context. Instead of the genes coding for cellulosomal proteins, M. pectinilyticus genome possessed 42 genes encoding extracellular proteins that were predicted to attach to the bacterial cell surface via S-layer homology (SLH) modules. Proteins were considered to contain SLH modules if SLH domains were identified by using both the dbCAN database and BlastP against the GenBank database. Among the SLH proteins of M. pectinilyticus, eight are associated with pectin-degrading CAZymes including PL1, CE8, CE12, and GH97; five contain peptidases; two contain N-acetylmuramoyl-L-alanine amidase; four contain domains with poorly characterized functions; and 23 have few identifiable domains other than SLH modules (Mw of

42 kDa – 318 kDa) (Figure 4.10). Searching the 23 uncharacterized SLH proteins and B9O19_241 against various protein databases (UniProt/SwissProt, Pfam, TIGRfam, and InterPro-entailed

databases) based on the protein sequence homology revealed that some SLH proteins contained little or no verifiable domains from which functional prediction could be made. SLH proteins were also searched against the GenBank database using the BlastP function. The GenBank protein database operated by NCBI contains a vast amount of peptide sequence data ranging from thoroughly characterized proteins to hypothetical proteins with no known functions, protein sequences from complete and draft quality bacterial genomes, as well as shotgun metagenome sequencing data of uncultured organisms. Proteins were considered to have found homologous matches in the GenBank protein database if the aligned proteins passed the minimum BlastP thresholds of 1) > 200 bit-score; or 2) < 200 bit-score, but > 20 % sequence coverage and > 40 % sequence identity. Among the 23 uncharacterized SLH proteins, only B9O19_1224 and B9O19_2167 found homologous matches which met the minimum requirements. B9O19_1224 showed ~ 500 bit-score, 88 % query cover, and 41 % identity to ‘S-layer domain-containing protein’ and ‘hexagonal wall protein’ from a Firmicutes bacterium CAG:41 (accession number CDB95905.1) and an uncultured Clostridium sp. (accession number SCH67055.1), respectively. B9O19_2167 showed ~ 500 bit-score, 98 % query cover, and 41 % sequence identity to a hypothetical protein from Sedimentibacter sp. B4 (accession number WP_019228207.1). While these matches should be considered as proteins of significant sequence similarity, no functional information could be deduced from the results. Therefore, it was concluded that all available search options were exhausted, and that the SLH proteins of M. pectinilyticus belong to previously undiscovered groups of proteins with potentially novel characteristics, structures, and functions. All but one (B9O19_1870) SLH proteins possessed N-terminal type I signal peptide, suggesting these proteins were secreted to the bacterial cell surface. B9O19_1870 contained a lipoprotein type II signal peptide. An in silico analysis of protein secondary structure using SPIDER2 predicted that the SLH proteins mostly consisted of a β-sheet profile spanning the majority of the protein regions, while the α-helix-rich regions of SLH proteins usually overlapping with CAZyme domains, dockerin-like domains, or SLH modules (Heffernan et al., 2016). The 23 uncharacterized

Figure 4.10 The 42 SLH module-containing proteins of M. pectinilyticus. Protein lengths are drawn to scale. Amino acid positions are indicated with numbers. Predicted protein domains are shown as rectangles, and coloured according to the domain category shown in the key. Where possible, additional domain details are labelled within domain rectangles. Remaining regions, with no known homology to existing protein domains, are represented by lines.

SLH proteins were aligned using ClustalW and BlastP to compare the protein sequence similarity (Figure 4.11). The most significant sequence similarities were observed between B9O19_50 and B9O19_1579, and between B9O19_1578 and B9O19_1579. While B9O19_1578 and B9O19_1579 had similar protein domain compositions made of SLH modules and putative type I and type II cohesion-like domains, respectively, B9O19_50 contained no known protein domains apart from the SLH repeats present at the C-terminal. The duplicated segments of type I cohesin-like domains on B9O19_1578 showed a high degree of protein sequence homology (74 %) to each other. B9O19_611 shared 35 % and 33 % sequence identities with B9O19_1545 and B9O19_1870, respectively. Overall, with few exceptions, SLH proteins lacked significant sequence homology to each other, suggesting

Figure 4.11 Percentage identity matrix of SLH domain-containing proteins of M. pectinilyticus. Protein sequences were aligned using ClustalW (Larkin et al., 2007) which expressed the protein identity in percentage format. Colour intensity corresponds to the sequence similarities relative to 100 % sequence identity which suggests perfect amino acid matches are found along the entire length of alignments.

that either these proteins were either not related in structures or functions, or the primary amino acid sequences of these proteins may not be the determining factor for shaping their 3-dimensional architecture.