Rumen protozoa

Rumen protozoa are reported to account for up to 50% of the microbial biomass in the rumen, and it has long been suspected that certain species play a significant role in the breakdown of plant material. There has been uncertainty, however, whether protozoa pro­duce their own cellulases and xylanases, or whether the activities that can be measured for the protozoal fraction of rumen contents are due to ingested bacteria. Rumen protozoa cannot currently be maintained pure culture. To resolve this issue, cDNA libraries have been constructed from polyA+ RNA (presumed to be of predominantly eukaroytic origin) from a protozoal-enriched fraction of rumen contents. In order to focus on individual proto­zoal species, material for extraction was initially obtained from sheep that carry a complete bacterial flora, but that had been de-faunated and mono-associated with single strains of protozoa. Screening for activity allowed the recovery of multiple cDNA clones that express CMCase or xylanase activity from Polyplastron multivesiculatum (76-78). Consistent with their protozoal origin, these genes show extremely biased codon usage, and AT-rich flanking regions, and fail to cross-hybridize with DNA of bacterial origin. Representatives of family 11 and 10 xylanases from rumen protozoa are generally reported that have rather simple domain structures, although the family 10 domain in the enzyme XynB is adjacent to a family 22 substrate-binding module (78).

Polysaccharidases in rumen protozoa are assumed to be expressed mainly within the food vacuoles, rather than being extra-cellular as in rumen bacteria and fungi. What con­sequences this has for their organization remains unknown, but it perhaps makes it more likely that they exist as soluble enzymes rather than as a cell-bound complex. Expression of

Neocallimastix patriciarum XynA (1) Neocallimastix patriciarum XynA (2)

Aspergillus niger XynA _ T richoderma reesei Xyn1

Aspergillus niger XynB
— Bacillus subtilis XynA

Streptomyces lividans XynA Streptomyces lividans XynB T richoderma reesei Xyn2

Bacillus pumilus XynA

Ruminococcus flavefaciens XynA Ruminococcus flavefaciens XynB Ruminococcus flavefaciens XynD Fibrobacter succinogenes XynC (1)

Fibrobacter succinogenes XynC (2)

Figure 12.2 Phylogenetic relationship between a xylanase from the rumen protozoan Polyplastron mul­tivesiculatum and other representative GH family 11 catalytic domains (based on Devillard et al. (77)). Sequences from rumen bacteria (Fibrobacter, Ruminococcus) and fungi (Neocallimastix) are shown in bold;those from non-rumen microorganisms are shown in normal type.

recombinant protozoal genes in E. coli maybe reduced because of their highly biased codon usage.

The sequence relationships of protozoal glycoside hydrolases show that they are often quite closely related to bacterial enzymes (77, 79) (Figure 12.2). They are, therefore, strong candidates for acquisition by horizontal gene transfer. The dense, mixed communities of the gut create obvious potential for such transfer events, particularly in the case of protozoa that are constantly engulfing and digesting rumen bacteria.