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Appendix A
Methods Involved in Modifying Rumen Bacteria

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:—

For the next step there are then two alternative approaches:—

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been prepared by combining parts of a Butyrivibrio plasmid with parts of an E. coli plasmid (Gregg, K. and Ware, C., unpublished).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:—

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:—

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 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:—

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.

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