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R. J. WallaceRuminal microbiology, biotechnology, and ruminant nutrition: progress and problems

1994, 72:2992-3003.J ANIM SCI

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Ruminal Microbiology, Biotechnology, and Ruminant Nutrition:Progress and Problems1R. J. Wallace

Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, U.K.

ABSTRACT: Present methods fo r manipulating ru-minal fermentation that involve microbial biotechnol-ogy include dietary ionophores, antibiotics, andmicrobial feed additives. Developments in recom-binant DNA technology mean t ha t future methods willhave a much wider scope. It has been suggested thatgenetically engineered ruminal microorganisms willbe used in future t o improve ruminal fermentation.Several technical objectives must be achieved beforethat will be possible. First, methods fo r insertingforeign o r modified genes into ruminal microorgan-isms and ensuring their efficient expression must bedeveloped. Broad host range plasmids and transposonshave been used successfully t o introduce new DNAinto ruminal bacteria, as have shuttle vectors con-structed as chimeras of plasmids from ruminal speciesand Escherichia coli. Although so far only antibioticresistance markers have been transferred, theprospects for introducing other genes into selectedruminal bacteria are excellent. Second, the expressionof the gene products(s) should be known t o benutritionally useful in vivo. A few examples of thistype of benefit have been demonstrated, and many

more proposed, including polysaccharidases for improving fiber digestion, methods for improving theamino acid composition of ruminal bacteria, andbreakdown of plant toxins. Third, the difficulty thathas been examined least, yet may prove most difficult o overcome, is that mechanisms have to be found fointroducing and maintaining the new strain in themixed ruminal population. Factors . governing thesurvival of new strains in vivo a re ill-understood, andattempts to select in favor of added new organismshave so far been unsuccessful. Because of the lasobstacle, it may be advantageous, at least in the shorterm, t o use nonruminal organisms, such as Saccharomyces cereuisiae, rather than indigenous ruminaspecies as a vehicle for implementing the benefits orecombinant DNA technology t o ruminal fermentation. Yeast is already in widespread use as a feedadditive, so no enrichment is necessary; and itsgenetics are already well known. Alternatively, addingparticular enzymes to the diet may achieve some othe objectives described above, with the advantagethat the manipulation could be achieved without therelease of a recombinant microorganism.

Key Words: Rumen, Ionophores, Biotechnology, Probiotics, Yeasts, Enzymes

IntroductionSince the development more than 40 yr ago of the

Hungate technique, by which the strictly anaerobicruminal bacteria could be cultivated for th e first time(Hungate, 19501, ruminal microbiology has had asignificant impact on microbiology in general. Hun-gates appreciation of the need to simulate the na turalecology of microbial ecosystems and his demonstrationof strict anaerobiosis opened up the entire area ofanaerobic microbiology. The nature of methanogenic

Presented at a symposium titled Cu rrent Aspects of Microbiol-ogy in th e Digestive Tract at the ASAS 85th Annu.Mtg., Spokane,WA.

Received November 5, 1993.Accepted June 28, 1994.

J. Anim. Sci. 1994. 72:2992-3003

bacteria and the concepts of interspecies H2 transfeand syntrophy whereby, for thermodynamic reasonsonly mixed cultures can carry out certain anaerobictransformations, were explored by Hungate, BryantWolin, and Wolfe (Smith and Hungate, 1958; annottet al., 1973;Tzeng et al., 1975;McInerney et al. , 1979Wolin and Miller, 1988) mainly using ruminamicroorganisms. More recently, a new class of fungiwhich had previously been mistaken for flagellateprotozoa, was discovered by Orpin in ruminal fluid(Orpin 1975, 19761, nd thi s discovery has generatedgreat interest in the distribution and function oanaerobic fungi. The rumen was additionally an ideaecosystem for studying microbial consortia attached t obiological surfaces (Cheng and Costerton, 1980).Understanding the microbiological transformationsthat occur in the rumen has also explained muchabout the nature of ruminal fermentation and the way

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RUMINAL MICROBIOLOGY AND BIOTECHNOLOGY 2993it affects ruminant nutrition. The physiological impor-tance of VFA production by the ruminal microorgan-isms to the nutrition of the host was established earlyon (Barcroft et al ., 1944); the mechanisms by whichthe fermentation acids are produced are for the mostpart similar t o those established for nonruminalbacteria (Russel l and Wallace, 1988).A few species ofbacteria, protozoa, and fungi carry out cellulolysis,and a more diverse population can hydrolyse starchand sugars (S tewa rt and Bryant, 1988). Protein iswasted by hydrolysis in the rumen because theruminal microorganisms use amino acids for energyproduction as well as for protein synthesis (Brodericket al., 1991; Russell e t al. 1991 ). Ciliate protozoa arenot essen tial for the fermentation, but their activitymay be considered beneficial o r detrimental t o nutri-tion depending on the animals nutritional status(Williams and Coleman, 1988). There are many otherexamples whereby an appreciation of microbiologicalevents in the rumen has influenced nutritionalpractices. Ruminal microbiology ha s also been impor-tant in providing concepts and quantitative dataessential for the construction of mathematical modelsthat predict ruminal function and ruminant nutrition(Baldwin and Koong, 1980; Russell et al., 1992).Microbiological explanations have proved valuable inavoiding digestive dysfunction, whether due t o as-sociative effects between fibrous and concentrate feeds(Stewart, 1977; Russell and Dombrowski, 19801,excessive lactate production (Dawson and Allison,19881, or bloat (Dawson and Allison, 1988).

Research in ruminal microbiology has therefore hada major impact in our understanding of ruminantnutrition. However, direct manipulation of ruminalfermentation by biotechnological means has so farbeen restricted t o a few antimicrobial compounds andsome microorganisms that can be added t o the feed.The rapid development of recombinant DNA technol-og y prompted several authors in the 1980s t o predictth at the new technology would be useful in developingnew str ains of ruminal bacteria that would benefit thenutrition of the host animal (Smith and Hespell,1983; Teather, 1985; Forsberg e t al., 1986; Hespell,1987, 1989; Russell and Wilson, 1988). The aims ofthis paper are to summarize briefly our understandingof present methods of manipulation on ruminalfermentation, t o review the progress that has beenmade in biotechnology relating t o ruminal microorgan-isms, and t o assess the likelihood of nutritionalbenefits ar ising from these advances in the foreseeablefuture.

Manipulation of Ruminal Fermentation byMicrobial and Antimicrobial Feed AdditivesIonophores and Antibiotics. Monensin is an iono-phore that was first used as a coccidiostat in poultry

and was then applied to ruminants from the mid-

1970s onward. It provides an economic benefit interms of feed efficiency (a n average 7.5% improve-ment; Goodrich et al., 19841, at least partly via itseffect on ruminal fermentation. Several other iono-phores have been identified that provide similarbenefits, including lasalocid, salinomycin, lysocellin,narasin, and tetronasin, and the peptide antibioticavoparcin is also effective. The mode of action of thesecompounds provoked much interest and research.

The toxicity of ionophores stems from their abilityto translocate ions across biological membranes andconsequently t o disrupt transmembrane ion gradients(Bergen and Bates, 1984; Russell and Strobel, 1989).Not all microorganisms are affected by ionophores:monensin and similar ionophores inhibit Gram-posi-tive bacteria more than Gram-negative bacteria(Chen and Wolin, 1979; Henderson et al., 1981;Nagaraja and Taylor, 1987). This selectivity is centralt o their manipulative effect, and depends on thepermeability of the cell envelope (cell wall and outermembrane in Gram-negative bacteria, cell wall inGram-positive bacter ia). This is why the antibioticavoparcin, which has a similar spectrum of antibac-terial activity but which kills sensitive cells byblocking cell wall synthesis, has a similar manipula-tive effect (St ew ar t et al., 1983). Therefore, toxicityand selectivity have different mechanisms, although ofcourse both are required for efficacy. Adaptation toionophore resistance, both of Gram-positive and ofresistant Gram-negative species such as Prevotellarumin ico la , is increasingly being realized t o play animportant part in the mode of action of ionophores too(Chen and Wolin, 1979; Henderson et al., 1981; deJong and Berschauer, 1983; Dawson and Boling, 1984;Kobayashi et al., 1989; Morehead and Dawson, 1992;Newbold et al., 1992) . Protozoa and fungi are alsosensitive t o ionophores to different extents, and it isnot clear how important these effects are compared tothe antibacterial effects (Dennis et al., 1986; Elliott etal., 1987; Newbold et al., 1988).

The effects that ionophores have on fermentation,such as changed fermentation stoichiometry andimproved protein flow from the rumen, are in manyways consistent with their effects on the bacterialpopulation (C he n and Wolin, 1979; Nagaraja andTaylor, 1987; Russell and Strobel, 1989). It has beensuggested that some effects, including changes inpropionate production, may be due primarily toantifungal rather than t o antibacterial effects (Ellio ttet al., 1987). Therefore, some ambiguity still existsconcerning the antimicrobial effects of ionophoresnearly 20 yr after their introduction. Furthermore, itis by no means clear whether the benefits of iono-phores stem from their effect in the rumen, or whethersome are postruminal in origin. Rogers et al. (1991)concluded that the effects of monensin on intestinaldigestion were minor. Nevertheless, they notedchanges in plasma glucose concentration when monen-sin was infused into the duodenum that could not be

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2994 WALLACEattributed to alterations in ruminal VFA profiles. I t i swell known that monensin affects mammalian as wellas microbial cells (e .g. , Shier and Dubourdieu, 1992).Microbial Feed Additives. Live microbial culturesand their extracts, particularly of Aspergillus oryzaeand Saccharomyces cereuisiae, have been used as feedadditives for many years. Their widespread use asmanipulating agents for ruminal fermentation, so -called direct-fed microbials, is more recent, as aremost of the research papers (original references canbe found in these reviews: Dawson 11990, 19921;Martin and Nisbet [19921; Wallace and Newbold[19921). On average, published data indicate thatmicrobial additives may benefit ruminant nutrition( in terms of live weight gain and milk production) bya similar magnitude to ionophores ( 7 or 8% improve-ment; Wallace and Newbold, 19931, in this case byincreasing feed intake rather than feed efficiency(Williams and Newbold, 1990). The effects are highlyvariable, however, and much remains to be estab-lished about the dose- and diet-dependence of theeffects.Different schemes have been drawn up by differentauthors to draw together into a logical mode of actionthe various observations that have been made onmicrobial feed additives (Williams, 1989; Offer, 1990;Wallace and Newbold, 1992). Figure 1 reflects ourmost recent findings and attempts to form theobservations into a sequence. The improved feedintake seems to be driven partly by an improved ra te(b ut usually not ext ent ) of fiber breakdown (Ma rtinand Nisbet, 1992; Wallace and Newbold, 1992) andpartly by an improved duodenal flow of absorbableamino-nitrogen (Williams et al., 1990; Erasmus e t al.,1992) . These two observations are suggested to arisefrom a more active microbial population: the mostreproducible effect of microbial feed additives is thatthey increase the viable count of anaerobic bacteriarecovered from ruminal fluid. Increases of 50 to 100%are common (Wallace and Newbold, 19931, butincreases of more th an 10-fold compared with controlshave been observed (Dawson et al., 1990) . Cellulolyticbacterial numbers are increased (Martin and Nisbet,1992; Wallace and Newbold, 1993) and lactic acid-utilizing bacteria are stimulated by the dicarboxylicacids present (Nisbet and Martin, 1990, 1991, 1993;Martin and Nisbet, 19921, thus explaining in par t theimprovement in fiber breakdown and increased stabil-ity of the fermentation in animals receiving yeast andA. oryzae (Harrison et al., 1988; Williams et al.,1991).

The questions then arises, Why should there besuch a stimulation in the viable count? Does it reflecta larger number of bacteria present, or that theproportion of culturablehiable organisms in the popu-lation increases? Also, what is the mechanism ofstimulation? Changes in the total protein concentra-tion in ruminal fluid are minor ( C J. Newbold and R.J. Wallace, unpublished results), so the increases

I Improved productivity+

Increased feed intakefIncreased rate of Increased flow ofcellulolysis microbial protein

Changed VFAproportionsfDecreased lactateproduction\ INCREASED BACTERIALVIABILITY

JAlteredmethanogenesis Improved pHstability

cemoval of oxygenby S. cerevisiaeFigure 1. A scheme describing the mode of action ofyeast culture.

presumably reflect a bacterial population that is nonecessarily greater in total but which has a higheratio of 1ive:dead cells. The mechanism depends on thefact that heat-labile-autoclaved yeast or A. oryzae losetheir ability to stimulate the viable count (Dawson eal., 1990; Newbold e t al., 1991). Thus e ither ametabolic activity or a heat-labile nutrient must beresponsible for the stimulatory activity. Dicarboxylicacids, which stimula te lactate uptake by Selenomonasr u m i n a n t i u m in vitro (Nisbet and Martin, 1990, 199119931, would be expected to survive autoclaving, aswould several other suggested mechanisms for yeasaction (Rose , 1987; Williams, 1989; Offer, 1990Martin and Nisbet, 1992). Recent work with differenstrains of yeast and respiration-deficient yeast mutants demonstrates that the ability of yeast tostimula te the viable count in the rumen depends on itrespiratory activity (Newbold et al., 1993). It isproposed that yeast removes some of the 0 2 thaoccurs in ruminal fluid at various times during the

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RUMINAL MICROBIOLOGY AND BIOTECHNOLOGY 2995daily feed cycle (Hillman et al., 1985 ) and , therefore,prevents toxicity t o the ruminal anaerobes. Lessattention has been focused on the precise mode ofaction of A. oryzae, but again, the activity is destroyedby autoclaving but not by irradiation (Newbold et al.,1991). Thus, either a metabolic activity o r a heat-labile nutrient must be responsible for the stimulatoryactivity. A. oryzae extract differs from yeast culture inth at the viable count of fungal cells is low (Newbold etal., 1991).

Regardless of the efficacy or mode of action ofmicrobial feed additives, they are already in wide-spread use. They may, in addition, offer new opportu-nities for manipulation. Dawson (199 2) has isolatedstrains of yeast th at stimulate the growth of specifictypes of bacteria, thus leading the development ofadditives suitable fo r different dietary circumstances.It is a relatively small step from there to introducenew activities into yeast that have specific and novelmodels of action, as will be described below.

Introduction of Genetically ModifiedMicroorganisms in the RumenRequirements . If genetically engineered ruminal

microorganisms are to be used for nutritional pur-poses, three scientific objectives must be met (Wal-lace, 1992) . The first is t o insert new genetic materialinto ruminal species and ensure that it is expressed,the second is to select a gene product or products thatwill benefit the nutrition of the host animal, and thethird is to establish a means by which the neworganism can survive. Aside from these aspects areethical considerations and regulatory constraints,which are complex and changing. They will not bediscussed here. Researchers must first establishwhether the objectives are achievable.Cloning of Genes fvom Ruminal Microorganisms.Since the first report of the successful cloning ofcellulases from Fibrobacter succinogenes into E . coli byCrosby et al. (19 84) , there have been many reports ofgenes being cloned from ruminal microorganisms intoother expression systems. Hespell ( 198 9) summarizedthe studies up to that time. Many more papers havebeen published since 1990; these are listed in Table 1,along with the genes cloned and the vector systemsused.

Several patterns become clear from examination ofHespell (1989) and Table 1: 1) The vectors andexpression are predominantly based on common E . colisystems, such as pUC-related plasmids, h phage, o rcosmids. Notable exceptions are the experiments byWhitehead and Hespell (19 90 ) and Whitehead et al.( 19911, who used Bacteroides frag ilis, B. uniformis ,and B. thetaiotaomicron expression systems. A Bac-teroides cloning system might be expected t o besuperior t o the E . coli ones for cloning genes fromruminal bacteria. The Bacteroides are more closely

related to ruminal organisms than E. coli an dtranslational and post-translational factors would beexpected to be more similar. 2) Genes have beencloned from many of the predominant species ofruminal bacteria, but only recently from the ruminalanaerobic fungi, by Gilbert et al. (1992) and Xue etal. (1 992a ,b). Because the fungi are eukaryotes,cloning must be done via the isolation of mRNA andpreparation of cDNA. To date, no genes have beencloned from the other ruminal eukaryotes, the ciliateprotozoa. 3) The enzymes tha t have been cloned havebeen with a single exception exclusively glycosidasesor polysaccharidases, particularly xylanases and en-doglucanases. In part this reflects the importance tha tis attached to fiber digestion in the rumen, but thereare also other reasons. The cloned genes can be readilydetected using chromogenic substrates, and the en-zymes are relatively stable. In addition, advances arebeing made rapidly in this area in the field ofnonruminant research, with the result that manygenes and sequences are available for comparativepurposes, enabling fundamental aspects of the originsand evolution of the genes to be analyzed.

Genetic and sequence studies have revealed thatsome polysaccharidase genes have a simple structure(Hazlewood et al., 1990; Cavicchioli et al., 1991; Uttet al., 19911, and other have several domains withdifferent functions. Several of the endoglucanases andxylanases have multiple catalytic domains of the same(Gilbert et al., 1992; Xue et al., 1992a) o r differentcatalytic properties (Zhang and Flint, 1992; Flint etal., 1993). Other domains are known t o be involved inbinding to the substrate (McGavin and Forsberg,1989), whereas yet others a re sequences of unusualamino acid composition that may form hinges in thegene product (Gilber t et al., 1992; Zhang and Flin t,1992; Flint et al., 1993 ). The functions of othersequences are as yet unknown (Lin and Thomson,199 a) . Sequence homologies also establish the ances-tral origins of the different genes (Gilbert et al., 199 2)and the scope of the different enzyme families that areinvolved in fiber breakdown (Ohmiya et al., 1989;Berger e t al. , 1990; Lin e t al., 1990; Mannarelli et al.,1990; Matsush ita e t al., 1990; Cavicchioli et al., 1991;Cunningham et al., 1991; Lin and Thomson, 1991b;Utt et al., 1991; Whitehead and Lee, 1991; Gilbert etal., 1992; Vercoe and Gregg, 1992; Wang and Thom-son, 1992; Flint et al., 1993).These studies given fascinating insight into thevariety of enzyme activities necessary to digest plantfiber. The complex nature of the polypeptidespresumably reflects adapta tions tha t have taken placeto cope with the difficulties of hydrolyzing insolubleand sometimes anhydrous substrates. The work isparticularly useful when used in combination withenzymological studies in which cellulases andxylanases of ruminal bacteria seem t o be highlyheterogeneous (e.g., Lin and Thomson, 1991b).Genetic characterization generally indicates that there

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2996 WALLACE

E&0ma,Mcu0

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RUMINAL MICROBIOLOGYare fewer genes than bands on zymograms, suggestingthat the heterogeneity observed in the zymograms isdue i n part to differences in post-translational modifi-cation (Hu et al., 1993).

Work with other classes of enzymes is only begin-ning. The importance of N metabolism in the rumenhas not yet led t o genetic work other than the cloningof glutamine synthetase from B. fibrisoluens and itscharacterization as a type I11 enzyme typical of gutBacteroides (Goodman and Woods, 1993).It must be emphasized that cloning genes fromruminal organisms is extremely worthwhile, leadingto an understanding of the genes, enzymes, andgenetic regulation in ruminal organisms. Such fun-damental information is essential for progress to bemade. Cloning and characterizing genes from ruminalmicroorganisms does not lead directly to the objectiveof using recombinant techniques t o improve ruminalfermentation, however.Expression of Foreign DNA in Ruminal Bacteria .Construction of an improved, genetically engineeredruminal microorganism will depend on developingsuitable genetic systems for the introduction of newDNA into ruminal species. Progress in this area issummarized in Table 2 .

Teather ( 19 85) reported the first instance in whichforeign DNA was transferred into a ruminal microbialspecies. The promiscuous plasmid RP4 was introducedinto Butyriv ibrio fibrisoluens by conjugal transfer fromE. coli, the successful transfer being demonstrated bythe acquisition of ampicillin resistance by B . fibrisol-uens. Similar conjugal transfer of tetracycline resis-tance between strains of P. ruminicola was success-fully carried out by Flint e t al. (1988). They provedthat the antibiotic resistant was plasmid-mediated byshowing that all resistant cells had acquired a90.5-kbp plasmid pRRI4. Russell and Wilson (1988)used the B. fragilis R751 plasmid containing a pE5-2shuttle vector to transfer erythromycin resistance

AND BIOTECHNOLOGY 2997between E. coli and P. ruminicola. Hespell (1989)reported brief details of filter matings between Strep-tococcus faecalis and B. f ibr i sohens in which theplasmid pAMPl was transferred t o B. fibrisoluens,conferring erythromycin resistance t o this organism.The first demonstration of transposon-mediated trans-fer into ruminal species was by Hespell and White-head (199 a). Tetracycline resistance was transferredby filter-mating Enterococcus faecalis with B. fibrisol-uens. The chromosomal insert Tn916 of E . faecalis wasshown to insert at one o r more separate chromosomalsites for four different B. fibrisolvens strains, eachrepresenting different DNA-relatedness groups. Con-jugal transfer between two different ruminal species(i.e., with ruminal species as both donor and recipi-ent) was achieved first by Hespell and Whitehead(1991b). The self-mobilizing plasmid pAMPl and thetransposon Tn196 were conjugated from E. faecalis toStreptococcus b ovis. The S. bovis transconjugants werethen used as donors for matings with B . fibrisoluens.Successful transfer was obtained with both vectors,although the frequency of transfer of Tn916 was verylow at 10-7 or less.

Electrotransformation, o r electroporation, wherebycells that are subjected to a high electromagnetic fieldbecome susceptible to transformation, has made thetransfe r of foreign genes into ruminal microorganismsa great deal easier, particularly with larger plasmids.Thomson and Flint (1 98 9) first showed the effective-ness of this procedure fo r P. ruminicola, again withthe plasmid pRRI4. The method of choice in construct-ing vectors fo r use either in conjugal transfer o relectroporation is now to make chimeric shutt levectors from well-characterized E. coli o r Bacteroidesplasmids and endogenous plasmids of ruminal species.Such vectors have been effective in transfers betweenE. coli and P. ruminicola as well as differentBacteroides species (Shoemaker et al., 1991; Thomsonet al., 1992). A cryptic plasmid (pBfl) from B.

Table 2. Transfer and expression of genes in ruminal bacteriaAuthors Gene Rumen species MethodFlint et al. (1988)Russell and Wilson (1988)Thomson and Flint (1989)Hespell and Whitehead (1991a)Hespell and Whitehead (1991b)Shoemaker et al. (1991)Cocconcelli et al. (1992)Thomson et al. (1992)Ware et al. (1992)Whitehead (1992)Whitehead (1992)Bechet et al. (1993)

Tetracycline resistanceErythromycin resistanceTetracycline resistanceTetracycline, erythromycin resistanceTetracycline, erythromycin resistanceTetracycline, erythromycin resistanceErythromycin resistanceErythromycin, clindamycin resistanceAmpicillin resistanceChloramphenicol resistanceErythromycin resistanceMultiple antibiotic resistance

P. ruminicolaP, ruminicolaP. ruminicolaB. fibrisoluensS. bouisP. ruminicolaR. albusP. ruminicolaB . fibrisoluensB. fibrisolvensS. bouisP. ruminicola

Conjugation with broad host rangeConjugal transfer, shuttle vectorElectrotransformation with plasmidConjugation with transposonConjugation with transposonShuttl e vectorElectrotransformation with plasmidElectrotransformation, conjugationElectrotransformation, shuttle vectorElectrotransformation, shuttle vectorElectrotransformation, shuttle vectorConjugation, electrotransformation

plasmid

with shuttle vector

with shuttle vector

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2998 WALLACEfibrisolvens was combined with an E . coli pUC18plasmid and a B. frugilis clindamycin resistance geneto give a shuttle vector that can be used to transformboth E. coli and B. f ibrisolvens (Ware et al., 1992).The first successful transformation of a Ruminococcusspecies was achieved using a lactococcal vector pCK17and pSC22, which were electroporated intoR. albus byCocconcelli et al. (1992).

Phages offer different opportunities as vectors.Large DNA fragments may be cloned, and, if thephage is lytic, problems associated with the detectionof cloned genes and intact bacteria a re overcome whenthe bacteria are lysed during the natural course ofphage infection and replication. Many different mor-photypes of phages are present in ruminal fluid(Klieve and Bauchop, 1991). A temperate phage wasisolated from S. r u m i n a n t i u m (Lockington et al.,1988 ), whereas lytic phages were found in P. r u m i n i -cola (Klieve et al., 1991) and S. bouis (Klieve andBauchop, 1991). However, the use of these phages asvectors has not been described.

So far, only evidence of gene transfer has beendiscussed. To make genetically engineered ruminalmicroorganisms useful, the new genes will have to beintegra ted into the chromosome, as was done with B.thetaiotamicron (Whitehead e t al., 1991); they willhave to be transcribed efficiently; they may need to bealtered by post-transcriptional modifications; andespecially in the case of polysaccharidases they mayneed t o be secreted from the cell. Some of theseenzymes have signal peptide sequences (Hazlewood etal., 1990; Gilbert et al., 1992; Flint et al., 19931, butothers do not (Mannarelli et al., 1990; Wang andThomson, 1990; Cunningham et al., 1991; Lin andThomson, 1991a; Utt et al., 19911, and a cytoplasmicor periplasmic location of the gene product has beencommon in E. coli (Berger et al., 1990; Cavicchioli andWatson, 1991; Hu et al., 1991). The choice of speciest o carry the new property may also be made moredifficult by restriction endonucleases, which makeintroduction of foreign DNA into some species moredifficult than others (Lee e t a l., 1992; Morrison et al.,1992a,b; Vanat et al., 1993). Clearly much develop-ment remains t o be done, but the prospects forachieving the objective of producing genetically en-gineered ruminal microorganisms are excellent. Inprinciple, there seems no reason, given time andeffort, that genetic systems cannot be developed forruminal bacteria that can be as effective as thosedeveloped for the main expression species such as E.coli.Potentially Useful New Properties. Most of thereviews mentioned above (Smith and Hespell, 1983;Teather, 1985; Forsberg et al., 1986; Hespell, 1987,1989; Russell and Wilson, 1988) made comprehensivesuggestions of the new properties that might beintroduced into ruminal microorganisms and benefitruminan t nutrition. Although virtually all the molecu-

lar work has dealt with cellulases and xylanasesthere is an evolutionary argument that questionwhether the introduction of microbes with differencellulases will improve fiber breakdown. One mighexpect that if a new cellulase were t o be beneficial, iwould a lready have evolved; the more optimistic viewis that evolution of cellulolytic ruminal microorganisms is not complete. Whichever is true, there icertainly one instance, as pointed out by Russell andWilson (198 8) , in which cellulases could be insertedinto ruminal bacter ia and be expected to be beneficialIf cellulases were produced by organisms that aremore tolerant t o lower ruminal pH than the existincellulolytic organisms, fiber digestion might be expected t o improve in animals receiving a highconcentrate ration. Even then, from the knowledgethat we already have about the complexity of thepolypeptides involved in fiber breakdown, it seemlikely that several genes will be required. Theevolutionary argument may be wrong, however, andperhaps the single gene would markedly improvecellulolysis. It is important to find out by doing theappropriate experiments.

Ruminal protein metabolism is arguably a morwasteful process nutritionally, and one th at should beapproached by genetic techniques. Proteins, peptidesand amino acids are broken down in the rumendestroying much of their nutritive value (Wallace andCotta, 1988) . Many species and enzymes are involvedin proteolysis (Wallace and Cotta, 1988), but Pruminicola seems to be predominant in peptidebreakdown (Wallace and McKain, 1991; McKain eal., 1992) . Ionophores are partly effective in slowingammonia production, probably by a combination oremoving some key species of amino acid-fermentinbacteria (Russell et al., 1991) and altering thepeptidolytic and deaminative activity of others (Newbold et al., 1990). Nevertheless, no direct inhibitors oprotein or peptide hydrolysis have yet been discoveredCiliate protozoa are extremely active in engulfing anddigesting ruminal bacteria, and thus decrease the negrowth yield in the rumen (Demeyer and Van Nevel1979). Antiprotozoal f ac tor s would, therefore, also beuseful properties t o introduce t o improve proteinnutrition of ruminants.

Detoxification is another area that could be veryfertile for genetic manipulation. Gregg and Sharp(1 99 1) described the background and problems associated with introducing bacteria that metabolizfluoroacetate, a common toxic component of Austra lian plants. Given the number and variety of toxiplant components that exist (Singleton and Kratzer1969; Dawson and Allison, 1988), the potential heret o improve nutrition by genetic means must be high

Alternatively, the new organism might be used as apackaging mechanism for delivering enzymes or smalmolecules t o the lower gut. Improving the amino acidprofile of microbial protein t o increase, for example

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RUMINAL MICROBIOLOGY A N D BIOTECHNOLOGY 2999lysine and methionine content was discussed byTeather (1985) and remains a viable objective.Establishment of New Organisms in th e Rumen.The best example of the successful introduction of anew (b ut not genetically modified) organism in therumen was the introduction of bacteria t hat degraded3-hydroxy-4(1H)-pyridone ( DHP) into Australianruminants. These animals were unable to use Leu-caena leucocephala because the non-protein aminoacid, mimosine, was converted to DHP, but no further .The DHP then had goitrogenic effects in the host.However, when the animals were inoculated withruminal fluid from goats adapted t o consume L.leucocephala, the DHP was further metabolized, thusavoiding the toxicity of effects of DHP (Jones andMegarrity, 1986; Dawson and Allison, 1988). Also ofinterest are reports that certain species of ruminalorganism can be used t o inoculate the rumen t oenhance rumen function. For example, whenMegasphaera elsdenii was added intraruminally, alarge inoculum decreased the severity of acute acidosisin cattle and smaller doses enhanced recovery ofruminal pH (Robinson et al., 1993). Inoculation oflambs with cultures of the rumen fungus, Neocal-Zimastix frontalis, promoted rumen function and leadt o earlier weaning (Theodorou et al., 1990).

The prospective new property t o be introduced intoa modified ruminal organism may sometimes select infavor of the modified strain. The prospects fo rmaintaining such a strain would be good. For exam-ple, the organism described above carrying cellulaseactivity and t ha t was able t o grow at pH lower thanindigenous cellulolytic bacteria would actually have aselective advantage under conditions in which it wouldbe most useful (Russell and Wilson, 1988), namelywhen a mixed diet is fed, the pH falls below 6.0, andcellulolysis is compromised. Similarly, new strainsth at detoxify components of the diet, such as the DHPdegraders mentioned above, may be competitive,particularly if they gain energy from the metabolictransformation. However, many inoculations, particu-larly of some of the suggested types of modifiedruminal bacteria, would be of organisms tha t would atbest be neutra l with respect to selectivity (Russel l andWilson, 1988; Gilbert and Hazlewood, 1991; Gregg andSharpe , 1991). The evolutionary argument comes intoplay again: if a modified organism were t o benefit bothitself and its host, it would already have evolved;therefore, because it has not, it would be difficult t oestablish. Furthermore, genetic modifications maythemselves impose a metabolic burden on the hostorganism, making it less competitive than the wildtype. Russell and Wilson (1988) demonstrated thatthe growth rate of a recombinant P. ruminicolacarrying an erythromycin resistance plasmid was one-third lower than that of the wild type, a property thatcaused the resistance to be lost if the cells weretransferred more than three times in batch cultures

lacking the antibiotic.Results obtained by tracking individual strains of

bacteria in vivo, when no external selective pressurewas applied, have been mixed. Adams et al. (1966)found that the number of nonruminal bacteria addedt o the rumen declined rapidly in vivo. A DNA probewas developed by Attwood et al. (1988) t o track P.ruminicola B14 in rumen fluid in vitro and in vivo.The strain had a half-life of 9 h or more in vitro butwas lost in vivo at a r ate corresponding to a half-life ofless than 30 min. It was suggested that the rapid losswas due to a bacteriocin-like activity present inruminal fluid (Attwood et al., 1988). Flint et a l.(1 989) also found that a rifampicin-resistant strain ofBacteroides multiacidus was lost rapidly (49%/h),such that the population of less than l o 3 mL-l wasdetectable 100 h after inoculation. In contrast, a r i pS . ruminantiurn, strain F100, persisted in the rumenat approximately l o 6 mL-l for at least 30 d. This isnot a universal property of S. ruminant ium, however,because another strain (SS2/R5) was effectively lostwithin 24 h of inoculation, despite the antibioticresistance mutation in SS2/R5 causing only a 10%decrease in growth rate (Wallace and Walker, 1993).Finally, Hespell (1 98 9) cites work by S. D. Mathieson,C. G. Orpin, and A. Blix in which a strain of B .fibrisolvens isolated from a reindeer was establishedsuccessfully in a sheep at approximately lo 5 cellsmL-l. Clearly, such a small population as thoseobserved by Flint et al. (1989) and Mathieson et al.would be unlikely to make a significant impact onruminal fermentation, unless the gene product had asecondary effect on other species. It does seem,however, that there are factors governing the survivalof bacteria in rumina l fluid tha t we do not understand.The presence of bacteriophages, mycoplasmas, andbacteriocin-like toxins is well known (Hoogenraad etal. , 1967; Jarvi s, 1968; Orpin and Munn, 1974; Klieveand Bauchop, 1988). What is much less clear is theeffect these have on the survivability of differentstrains and on the metabolic inefficiency this maycause in ruminal fermentation.

It may be possible t o devise a selection strategywhereby an ecological niche is effectively created forthe new organism in vivo by adding certain materialsto the feed. Many sugars and sugar alcohols that canbe utilized by nonruminant species are degraded onlyslowly in rumen contents (Wallace, 1989). Experi-ments in which sorbitol-utilizingE . coli or S. ruminan-t i u m were inoculated into the rumen and sorbitol wasadded t o the feed as a selective energy source fo r thegrowth of these strains were unsuccessful. The E. colistrain was simply unsuited t o growth under ruminalconditions (Wallace et al., 19891, but the reason forthe failure of the S. ruminant ium strain t o survivewas, as before, unknown (Wallace and Walker, 1993).A further problem of using indigenous ruminalbacteria as vehicles fo r manipulating ruminal fermen-

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3000 WALLACEtation is their obligately anaerobic physiology. Growthand inoculation with a suitable culture, whilestraightforward in the anaerobic microbiology labora-tory, would not easily lend itself to farm practices,particularly if repeated inoculation proved necessary.Thus there are several potential problems associatedwith introducing and establishing modified ruminalorganisms that will grow and produce new geneproducts in vivo. Indeed, given the complexity of theproblems, solutions may be difficult t o find.

Alternatives to Ruminal Microorganisms forImplementing Molecular TechniquesOther than E. col i , the genetics of the yeast, 5'.cereuisiae, are among the best known of all biological

organisms (Johnston, 1988). Thus, any desiredgenetic modifications could be made more readily withS. cereuisiae than with ruminal microorganisms. Yeastis already used as a feed additive, as described above,so growth of the organism in vivo is not an issue. Livecells are supplied at each meal and although yeastcells grow only slowly, if at all, in the rumen, theyremain viable and metabolically active (Wallace andNewbold, 1992 ). The recombinant productfactivitywould then complement a pre-existing beneficialaction. I t is logxal, therefore, t o suggest that, a t leastin the short term, S. cereuisiae might be a moreappropriate vehicle than indigenous ruminal organ-isms for implementing the benefits of recombinantDNA technology in the area of ruminal fermentationand ruminant nutrition.

The release of recombinant organisms is the lastarea where problems would undoubtedly be encoun-tered, whether the organism were a ruminal species o rS. cereuisiae. It is difficult to forecast how regulatoryauthorities will view the use of a live recombinantmicroorganism entering the food chain in ruminants.An even more pragmatic approach might therefore bet o investigate, where the desired effect can beprovided by single gene products such as an enzyme,whether it may be possible to manufacture therecombinant enzyme in a fermenter and feed directly apurified extract containing the enzyme. The success ofsuch an approach would depend on the stability of theenzyme to breakdown by ruminal microorganisms,which might be difficult t o achieve. It would also onlybe applicable where extracellular enzymes, probablyhydrolases, rather than intracellular enzymes wereinvolved. However, the approach would have theadvantage that it would not require the release of arecombinant microorganism. Dietary enzymes havebeen used extensively in nonruminants (Chesson,19931, and indications are favorable that similarpreparations can be effective in ruminants (Konno etal., 1993; Dawson, 1993).

ImplicationsMolecular techniques must continue t o be used t o

determine the fundamental details of how ruminalmicroorganisms and their enzymes carry out digestionand fermentation. The benefits of this knowledge canonly be implemented, however, when other objectivesincluding the development of genetic systems inruminal microorganisms and the establishment ofthese modified organisms in vivo, have been resolved,and it may be advantageous in the short term t oconcentrate on using yeast or recombinant enzymesalone t o produce nutritional benefits. 'When geneticand establishment problems have been solved withindigenous ruminal bacteria, then the added advan-tages that they might confer, such as suitability inextensive systems, requiring single inoculationsrather than daily feeding, will be able t o be exploited.

Literature CitedAdams, J. C., P. A. Hartman , and N. L. Jacobsen. 1966. Longevity of

selected exogenous microorganisms in the rumen. Can. J.Microbiol. 12:363.

Attwood, G. T., R. A. Lockington, G.-P. Xue, and J. D. Brooker. 1988Use of a unique gene sequence as a probe t o enumerate a strainof Bacteroides ruminicola introduced into the rumen. Appl.Environ. Microbiol. 54:534.

Baldwin, R. L., and L. J. Koong. 1980. Mathematical modelling inanalyses of ruminant digestive function: Philosophy, methodol-ogy and application. In: Y. Ruckebusch and P. Thivend (Ed.)Digestive Physiology and Metabolism in Ruminants. p 251MTP Press, Lancaster, UK.

Barcroft, J. , R. A. McAnally, and A. T. Phillipson. 1944. Absorptionof volatile acids from the alimentary tract of sheep and otherspecies. J. Exp. Biol. 20:120.

Bechet, M., P. Pheulpin, H. J. Flint, J. Martin, and H.-C. Dubour-gier. 1993. Transfer of hybrid plasmids based on the repliconpRR17 from Escherichia coli to Bacteroides an d Prevotellastrains. J. Appl. Bacteriol. 74:542.

Bergen, W. G., and D. B. Bates. 1984. Ionophores: Their effect onproduction efficiency and mode of action. J. Anim. Sci. 58:1465

Berger, E., W. A. Jones, D. T. Jones, and D. R. Woods. 1990Sequencing and expression of a cellodextrinase ( cedl) genefrom Butyriuibrio fibrisolvens H17c cloned in Escherichia coliMol. Gen. Genet. 223:310.

Broderick, G. A,, R. J. Wallace, and E . R. 0rskov. 1991. Control ofrat e and ex tent of protein degradation. In: T. Tsuda, Y. Sasakian d R. Kawashima ( Ed. ) Physiological Aspects of Digestionand Metabolism in Ruminants . p 541. Academic Press, London

Cavicchioli, R., P. D. East, and K. Watson. 1991. endAFS, a novelfamily E endoglucanase gene from Fibrobacter succinogenesARl. J . Bacteriol. 173:3265.

Cavicchioli, R., and K. Watson. 1991. Molecular cloning, expression,and characterization of endoglucanase genes from Fibrobactersuccinogenes AR1. Appl. Environ. Microbiol. 57:359.

Chen, M., and M. J. Wolin. 1979. Effect of monensin and lasalocid-sodium on the growth of methanogenic and rumen saccharolytic bacteria. Appl. Environ. Microbiol. 385'2.

Cheng, K-J., and J. W. Costerton. 1980. Adherent rumenbacteria-Their role in the digestion of plan t material, u rea andepithelial cells. In: Y. Ruckebusch and P. Thivend (Ed.) p 227MTP Press, Lancaster, UK.

Chesson, A. 1993. Feed enzymes. Anim. Feed Sci. Technol. 4565Cocconcelli, P. S., E. Ferrari, F. Rossi, and V. Bottazzi. 1992. Plas

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Cotta, M. A., an d T. R. Whitehead. 1993. Regulation and cloning ofthe gene encoding amylase activity of the ruminal bacteriumStreptococcus bovis. Appl. Environ. Microbiol. 59:189.

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