Although there are a number of groups around the world that are already researching the possibilities of modifying rumen bacteria most research is in its preliminary phase. The general methods are given in outline below:—
Isolation of Genes for Fibrolytic Enzymes
These usually follow the following course:—
the first step follows standard methods for extraction of genomic DNA from fibre digesting bacteria (and attempts are also being made with fungi)
the DNA is fragmented by partial digestion with restriction endonucleases and followed by selection of suitably sized fragments using gel or gradient fractionation. (For plasmid libraries a suitable size is 3–8 kilobases (kb) or 30–40 kb for cosmid libraries)
For the next step there are then two alternative approaches:—
the DNA fragments are ligated to appropriate vectors such as pUC.18 or pUC.19 plasmids and the recombinant DNA can then be used to transform E. coli. An alternative is to ligate fragments to cosmid vectors, package the DNA into lambda phage particles and transfect into host E. coli
recombinant colonies will then be selected for their ability to digest cellulose, hemicellulose or xylan. For cosmids, fragments of inserted DNA require sub-cloning into plasmids for closer analysis and these must be re-screened for enzyme activity in the plasmid subclones
the subcloned fragments are “trimmed down” by using restriction enzymes to delete DNA fragments progressively from the ends of the isolated DNA. In this way the limits of the gene should be defined
the “trimmed down” recombinant genes will then be sequenced by the Sanger “dideoxy” chain-termination procedure (Sanger et al., 1977)
once the genes have been defined, they may be excised for insertion into an anaerobic bacterial plasmid.
Preparation of Anaerobic Bacterial Plasmids Suitable for Gene Insertion
A shuttle plasmid has proven to be the most appropriate vector. This is because transformation of rumen anaerobes is likely to be relatively inefficient in the early stages and the ability to grow large quantities of plasmid in E. coli may be essential. The first plasmid has been prepared by combining parts of a Butyrivibrio plasmid with parts of an E. coli plasmid (Gregg, K. and Ware, C., unpublished). The essential features included in this are as follows:—
an E. coli origin of replication and antibiotic resistance gene
an origin of replication and antibiotic resistance gene active in the anaerobic host
a multiple cloning site, cleavable by a series of restriction endonucleases for insertion of genes
manageable size of the recombinant plasmid (e.g. 5–6 kb) to ensure optimum transformation capability, with minimal risk of DNA deletion or rearrangement within the anaerobic host.
Location of Gene Control Factors Located Externally to the Enzyme-Coding Sequences
For this purpose it will be necessary to test cloned genes for suppression by simple carbohydrate nutrients. Where this feature is an intrinsic part of the enzyme gene, additional control sequences may be necessary. However, it is possible that separate DNA fragments from the original donor genome may be required to provide a workable control system. Furthermore it is quite likely that gene control may require the combined action of multiple genes and at this stage the use of cosmids, with their capability for larger DNA fragment insertion, may be essential.
Integration of Introduced Genes into the Chromosome of the Host Bacterium
The likely approach here is as follows:—
a short segment of the host bacterial DNA will be introduced into the gene-bearing plasmid
the plasmid will be inserted into the host bacterium and the culture treated, with a physical or chemical agent, to induce recombination repair. Examples include the use of UV-light or chemicals such as ethyl methane sulphonate
the bacteria must then be grown under “curing” conditions to encourage loss of free plasmids from the bacteria. Suitable curing agents to test might be acridine orange or deoxycholate
to identify the organisms that have integrated the introduced DNA into their chromosomes, the bacteria must then be selected for retention of antibiotic resistance and for their ability to digest fibre, in the absence of plasmids.
Direct Transformation of Rumen Anaerobes Plasmids capable of replication in rumen anaerobes have already been identified (Teather, 1982) and some recombinant plasmids have been constructed for growth in human colonic anaerobes (Smith, 1985).
Problems that need to be studied are:—
the possibility that host bacteria may contain uncharacterised restriction endonucleases, capable of digesting any DNA introduced
the possibility that plasmids already constructed for growth in colonic species (e.g. Bacteroides fragilis) may not possess the features necessary for maintenance in rumen species of Bacteroides.
The predictable results of these problems are that the presence of uncharacterised restriction endonucleases may lead to extremely low transformation efficiency, although, once grown in a particular species, the plasmid will be protected for further transformation in that species. If the features necessary for maintenance of rumen species are absent from available plasmids, then this would lead to complete failure of the plasmid to grow and may necessitate locating naturally occurring plasmids in rumen bacteria, to obtain suitable replication control sequences.
The Requirements for Research once Recombinant Rumen Bacteria are Developed
There seems to be every optimism, with the rapid development of molecular genetics, that new strains of rumen micro-organisms will be developed with enhanced capabilities to digest (ferment) fibre. The required capabilities are not entirely clear but include microbes with the following characteristics:—
enhanced enzyme production
multi-enzyme systems allowing new strains to cope with a wider range of nutritive substrates (e.g. a largely pectin fermenter may be given the ability to digest cellulose and hemicellulose)
multi-enzyme systems for the uptake of ammonia; this could be extremely important for survivability of bacteria. If a bacterium could switch enzymes to adapt to changing ammonia levels, this would effectively protect its survival in the rumen and may give it a competitive edge, particularly if it could also use a number of carbohydrate substrates
possession of enzymes modified by protein engineering, which may allow more efficient function in vivo.
Establishment and growth of such organisms in culture should have few difficulties, since antibiotic resistance will also be cloned into them. However, the maintenance of these organisms in the rumen will be extremely difficult because of the interactive and competitive nature of the complex microbial ecosystem within the rumen.
Considerable research is necessary to develop techniques to examine the ecological niche of these organisms, their survivability, the factors involved in maintaining them in the rumen and their growth characteristics.
Monitoring the numbers of an individual species will become necessary in order to estimate their representation within the rumen biomass and their growth characteristics. Microbes in the rumen are generally in two major pools which reversibly exchange but which are not necessarily in equilibrium (i.e. the pool free in the liquid medium (the colonizing pool) and the pool of microbes attached to particles (the sequestered pool)). This will make the task of estimating the contribution of individual organisms quite difficult.
Isotope technology coupled with genetic engineering must be put into action to solve the problem. In preliminary work in our laboratory, 15N, 35S, 14C and 3H labelling of bacteria is being undertaken both in vivo and in vitro. The specific radioactivity time relationships of “labelled” microbes in the rumen are being followed in order to quantitate bacterial growth rate. Only mixed populations of rumen microorganisms are being examined at the present time, but the technology must eventually lead to labelling of individual organisms that are then returned to the rumen to label the pool of these organisms. To follow the dilution (mixing and therefore pool size and subsequent growth of the organism) individual species will have to be identified in mixed populations of rumen organisms. This may be achieved by developing DNA hybridization probes for individual rumen species.
Once the growth, turnover and survivability of the rumen microbes have been tested under laboratory conditions, then the laboratory technology must be adapted and tested under the conditions pertaining to the livestock producer.
