Genomic and enzymatic results show Bacillus cellulosilyticus uses a novel set of LPXTA carbohydrases to hydrolyze polysaccharides

Abstract

Background: Alkaliphilic Bacillus species are intrinsically interesting due to the bioenergetic problems posed by growth at high pH and high salt. Three alkaline cellulases have been cloned, sequenced and expressed from Bacillus cellulosilyticus N-4 (Bcell) making it an excellent target for genomic sequencing and mining of biomass-degrading enzymes.

Methodology/principal findings: The genome of Bcell is a single chromosome of 4.7 Mb with no plasmids present and three large phage insertions. The most unusual feature of the genome is the presence of 23 LPXTA membrane anchor proteins; 17 of these are annotated as involved in polysaccharide degradation. These two values are significantly higher than seen in any other Bacillus species. This high number of membrane anchor proteins is seen only in pathogenic gram-positive organisms such as Listeria monocytogenes or Staphylococcus aureus. Bcell also possesses four sortase D subfamily 4 enzymes that incorporate LPXTA-bearing proteins into the cell wall; three of these are closely related to each other and unique to Bcell. Cell fractionation and enzymatic assay of Bcell cultures show that the majority of polysaccharide degradation is associated with the cell wall LPXTA-enzymes, an unusual feature in gram-positive aerobes. Genomic analysis and growth studies both strongly argue against Bcell being a truly cellulolytic organism, in spite of its name. Preliminary results suggest that fungal mycelia may be the natural substrate for this organism.

Conclusions/significance: Bacillus cellulosilyticus N-4, in spite of its name, does not possess any of the genes necessary for crystalline cellulose degradation, demonstrating the risk of classifying microorganisms without the benefit of genomic analysis. Bcell is the first gram-positive aerobic organism shown to use predominantly cell-bound, non-cellulosomal enzymes for polysaccharide degradation. The LPXTA-sortase system utilized by Bcell may have applications both in anchoring cellulases and other biomass-degrading enzymes to Bcell itself and in anchoring proteins other gram-positive organisms.

Figure 1. Phylogenetic tree highlighting the position of Bacillus cellulosilyticus N-4 DSM 2522 within the Bacillales.

The strains and their corresponding NCBI accession numbers: Bacillus hemicellulosilyticus DSM 16731^T AB043846; Bacillus halodurans XJU-2 AY960851; Bacillus akibai ATCC 43226^T AB043858; Bacillus macyae DSM 16346^T AY032601; Bacillus alkalidiazotrophicus UNIQEM U377^T EU143680; Bacillus alkalinitrilicus UNIQEM U240^T EF422411; Bacillus algicola LS7 FJ937877; Bacillus mannanilyticus DSM 16130^T AB043864; Bacillus pseudofirmus SVB1 EU533950; Bacillus cellulosilyticus DSM 2522^T AB043852; Bacillus vedderi DSM 9768^T Z48306; Bacillus polygoni JCM 14604^T AB292819; Bacillus clarkia DSM 8720^T X76444; Bacillus agaradhaerens GSP78 AY553093; Bacillus aurantiacus B1-1 AJ606036; Bacillus amyloliquefaciens CMB01 AF489591; Bacillus subtilis AY887082; Bacillus licheniformis 11 FJ435222; and Bacillus alveayuensis KCTC 10634^T AY605232.

Figure 2. Bcell growth on A. niger mycelia.

Microscopic images (2000× magnification) of Bcell culture growing on A. niger mycelia; same field with transmitted light.

Figure 3. Bcell growth on A. niger mycelia.

Microscopic images (2000× magnification) of Bcell culture growing on A. niger mycelia; same field with dark field epifluorescence.