Ruminal Microbiology, Biotechnology, and Ruminant Nutrition: Progress and Problems1

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Ruminal Microbiology, Biotechnology, and Ruminant Nutrition:
Progress and Problems1
R.
J.
Wallace
Rowett Research Institute, Bucksburn, Aberdeen
AB2
9SB,
U.K.
ABSTRACT:
Present methods for manipulating ru-
minal fermentation that involve microbial biotechnol-
ogy include dietary ionophores, antibiotics, and
microbial feed additives. Developments in recom-
binant DNA technology mean that future methods will
have
a
much wider scope. It has been suggested that
genetically engineered ruminal microorganisms will
be used in future
t o
improve ruminal fermentation.
Several technical objectives must be achieved before
that will be possible. First, methods
for
inserting
foreign
or
modified genes into ruminal microorgan-
isms and ensuring their efficient expression must be
developed. Broad host range plasmids and transposons
have been used successfully
t o
introduce new DNA
into ruminal bacteria,
as
have shuttle vectors con-
structed as chimeras
of
plasmids from ruminal species
and
Escherichia coli.
Although
so
far only antibiotic
resistance markers have been transferred, the
prospects for introducing other genes into selected
ruminal bacteria are excellent. Second, the expression
of the gene products(s) should be known
t o
be
nutritionally useful in vivo. A few examples of this
type of benefit have been demonstrated, and many
more proposed, including polysaccharidases for im-
proving fiber digestion, methods
for
improving the
amino acid composition
of
ruminal bacteria, and
breakdown
of
plant toxins. Third, the difficulty that
has been examined least, yet may prove most difficult
t o
overcome, is that mechanisms have to be found for
introducing and maintaining the new strain in the
mixed ruminal population. Factors
.
governing the
survival of new strains in vivo are ill-understood, and
attempts to select in favor of added new organisms
have so far been unsuccessful. Because of the last
obstacle, it may be advantageous, at least in the short
term,
t o
use nonruminal organisms, such as
Sac-
charomyces cereuisiae,
rather than indigenous ruminal
species as a vehicle for implementing the benefits of
recombinant DNA technology
t o
ruminal fermenta-
tion. Yeast is already in widespread use as a feed
additive, so no enrichment is necessary; and
its
genetics are already well known. Alternatively, adding
particular enzymes to the diet may achieve some of
the objectives described above, with the advantage
that the manipulation could be achieved without the
release
of
a recombinant microorganism.
Key Words: Rumen, Ionophores, Biotechnology, Probiotics, Yeasts, Enzymes
Introduction
Since the development more than
40
yr
ago of the
Hungate technique, by which the strictly anaerobic
ruminal bacteria could be cultivated for the
first
time
(Hungate,
19501,
ruminal microbiology has had
a
significant impact on microbiology in general. Hun-
gate’s appreciation
of
the need to simulate the natural
ecology
of
microbial ecosystems and his demonstration
of strict anaerobiosis opened up the entire area
of
anaerobic microbiology. The nature
of
methanogenic
‘Presented at
a
symposium titled “Current Aspects of Microbiol-
ogy
in the 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
transfer
and syntrophy whereby, for thermodynamic reasons,
only mixed cultures can
carry
out certain anaerobic
transformations, were explored by Hungate, Bryant,
Wolin, and Wolfe (Smith and Hungate,
1958;
Iannotti
et al.,
1973;
Tzeng et al.,
1975;
McInerney et al.,
1979;
Wolin and Miller,
1988)
mainly using ruminal
microorganisms. More recently,
a
new class
of
fungi,
which had previously been mistaken for flagellate
protozoa, was discovered by Orpin in ruminal fluid
(Orpin
1975, 19761,
and this discovery has generated
great interest in the distribution and function of
anaerobic fungi. The rumen was additionally an ideal
ecosystem for studying microbial consortia attached
t o
biological surfaces (Cheng and Costerton,
1980).
Understanding the microbiological transformations
that occur in the rumen has also explained much
about the nature
of
ruminal fermentation and the way
2992
RUMINAL MICROBIOLOGY AND BIOTECHNOLOGY
2993
it affects ruminant nutrition. The physiological impor-
tance of VFA production by the ruminal microorgan-
isms to the nutrition of the host was established early
on (Barcroft et al., 1944); the mechanisms by which
the fermentation acids are produced are for the most
part similar
t o
those established for nonruminal
bacteria (Russell and Wallace, 1988).
A
few species
of
bacteria, protozoa, and fungi carry out cellulolysis,
and a more diverse population can hydrolyse starch
and sugars (Stewart and Bryant, 1988). Protein is
wasted by hydrolysis in the rumen because the
ruminal microorganisms use amino acids for energy
production as well as
for
protein synthesis (Broderick
et al., 1991; Russell et al. 1991). Ciliate protozoa are
not essential for the fermentation, but their activity
may be considered beneficial
or
detrimental
t o
nutri-
tion depending on the animal’s nutritional status
(Williams and Coleman, 1988). There are many other
examples whereby an appreciation
of
microbiological
events in the rumen has influenced nutritional
practices. Ruminal microbiology has also been impor-
tant in providing concepts and quantitative data
essential for the construction of mathematical models
that predict ruminal function and ruminant nutrition
(Baldwin and Koong, 1980; Russell et al., 1992).
Microbiological explanations have proved valuable in
avoiding 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 had
a major impact in our understanding
of
ruminant
nutrition. However, direct manipulation
of
ruminal
fermentation by biotechnological means has
so
far
been restricted
t o
a few antimicrobial compounds and
some microorganisms that can be added
t o
the feed.
The rapid development of recombinant DNA technol-
ogy prompted several authors in the 1980s
t o
predict
that the new technology would be useful in developing
new strains of ruminal bacteria that would benefit the
nutrition of the host animal (Smith and Hespell,
1983; Teather, 1985; Forsberg et al., 1986; Hespell,
1987, 1989; Russell and Wilson, 1988). The aims
of
this paper are to summarize briefly our understanding
of
present methods of manipulation on ruminal
fermentation,
t o
review the progress that has been
made in biotechnology relating
t o
ruminal microorgan-
isms, and
t o
assess the likelihood of nutritional
benefits arising from these advances in the foreseeable
future.
Manipulation
of
Ruminal Fermentation
by
Microbial and Antimicrobial Feed Additives
Ionophores
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 in
terms
of
feed efficiency ( an average 7.5% improve-
ment; Goodrich et al., 19841, at least partly via its
effect on ruminal fermentation. Several other iono-
phores have been identified that provide similar
benefits, including lasalocid, salinomycin, lysocellin,
narasin, and tetronasin, and the peptide antibiotic
avoparcin is also effective. The mode
of
action
of
these
compounds provoked much interest and research.
The toxicity
of
ionophores stems from their ability
to translocate ions across biological membranes and
consequently
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 central
t o
their manipulative effect, and depends on the
permeability of the cell envelope (cell wall and outer
membrane in Gram-negative bacteria, cell wall in
Gram-positive bacteria). This is why the antibiotic
avoparcin, which has a similar spectrum of antibac-
terial activity but which kills sensitive cells by
blocking cell wall synthesis, has a similar manipula-
tive effect (Stewart et al., 1983). Therefore, toxicity
and selectivity have different mechanisms, although of
course both are required for efficacy. Adaptation to
ionophore resistance, both of Gram-positive and
of
resistant Gram-negative species such as Prevotella
ruminicola,
is increasingly being realized
t o
play an
important part in the mode
of
action
of
ionophores too
(Chen and Wolin, 1979; Henderson et al., 1981; de
Jong and Berschauer, 1983; Dawson and Boling, 1984;
Kobayashi et al., 1989; Morehead and Dawson, 1992;
Newbold et al., 1992). Protozoa and fungi are also
sensitive
t o
ionophores to different extents, and it is
not clear how important these effects are compared to
the antibacterial effects (Dennis et al., 1986; Elliott et
al., 1987; Newbold et al., 1988).
The effects that ionophores have on fermentation,
such as changed fermentation stoichiometry and
improved protein flow from the rumen, are in many
ways consistent with their effects on the bacterial
population (Chen and Wolin, 1979; Nagaraja and
Taylor, 1987; Russell and Strobel, 1989). It has been
suggested that some effects, including changes in
propionate production, may be due primarily to
antifungal rather than
t o
antibacterial effects (Elliott
et al., 1987). Therefore, some ambiguity still exists
concerning the antimicrobial effects of ionophores
nearly
20
yr after their introduction. Furthermore, it
is by no means clear whether the benefits
of
iono-
phores stem from their effect in the rumen, or whether
some are postruminal in origin. Rogers et al. (1991)
concluded that the effects
of
monensin on intestinal
digestion were minor. Nevertheless, they noted
changes in plasma glucose concentration when monen-
sin was infused into the duodenum that could not be
2994
WALLACE
attributed to alterations in ruminal
VFA
profiles. It is
well known that monensin affects mammalian as well
as microbial cells (e.g., Shier and Dubourdieu, 1992).
Microbial Feed Additives.
Live microbial cultures
and their extracts, particularly of
Aspergillus oryzae
and
Saccharomyces cereuisiae,
have been used as feed
additives for many years. Their widespread use as
manipulating agents for ruminal fermentation, so-
called direct-fed microbials, is more recent, as are
most of the research papers (original references can
be found in these reviews: Dawson 11990, 19921;
Martin and Nisbet
[
19921; Wallace and Newbold
[19921). On average, published data indicate that
microbial additives may benefit ruminant nutrition
(i n terms of live weight gain and milk production) by
a similar magnitude to ionophores
( 7
or
8%
improve-
ment; Wallace and Newbold, 19931, in this case by
increasing feed intake rather than feed efficiency
(Williams and Newbold, 1990). The effects are highly
variable, however, and much remains to be estab-
lished about the dose- and diet-dependence of the
effects.
Different schemes have been drawn up by different
authors to draw together into a logical mode of action
the various observations that have been made on
microbial feed additives (Williams, 1989; Offer, 1990;
Wallace and Newbold, 1992). Figure 1 reflects our
most recent findings and attempts to form the
observations into a sequence. The improved feed
intake seems to be driven partly by an improved rate
(but usually not extent) of fiber breakdown (Martin
and Nisbet, 1992; Wallace and Newbold, 1992) and
partly by an improved duodenal flow of absorbable
amino-nitrogen (Williams et al., 1990; Erasmus et al.,
1992). These two observations are suggested to arise
from a more active microbial population: the most
reproducible effect of microbial feed additives
is
that
they increase the viable count of anaerobic bacteria
recovered from ruminal fluid. Increases of
50
to 100%
are common (Wallace and Newbold, 19931, but
increases of more than 10-fold compared with controls
have been observed (Dawson et al., 1990). Cellulolytic
bacterial numbers are increased (Martin and Nisbet,
1992; Wallace and Newbold, 1993) and lactic acid-
utilizing bacteria are stimulated by the dicarboxylic
acids present (Nisbet and Martin, 1990, 1991, 1993;
Martin and Nisbet, 19921, thus explaining in part the
improvement in fiber breakdown and increased stabil-
ity of the fermentation in animals receiving yeast and
A.
oryzae
(Harrison et al., 1988; Williams et al.,
1991).
The questions then arises,
Why
should there be
such a stimulation in the viable count? Does
it
reflect
a larger number of bacteria present, or that the
proportion of culturablehiable organisms in the popu-
lation increases? Also, what
is
the mechanism of
stimulation? 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 intake
f
Increased rate of Increased
flow
of
cellulolysis microbial protein
Changed VFA
proportions
f
Decreased lactate
production
\
I
INCREASED BACTERIAL
VIABILITY
J
Altered
methanogenesis
Improved pH
stability
c
Removal of oxygen
by
S.
cerevisiae
Figure
1.
A
scheme describing the mode
of
action
of
yeast culture.
presumably reflect a bacterial population that is not
necessarily greater in total but which has a higher
ratio of 1ive:dead cells. The mechanism depends on the
fact that heat-labile-autoclaved yeast or
A.
oryzae
lose
their ability to stimulate the viable count (Dawson et
al., 1990; Newbold et al., 1991). Thus either a
metabolic activity or a heat-labile nutrient must be
responsible for the stimulatory activity. Dicarboxylic
acids, which stimulate lactate uptake by
Selenomonas
ruminantium
in vitro (Nisbet and Martin, 1990, 1991,
19931, would be expected to survive autoclaving, as
would several other suggested mechanisms for yeast
action (Rose, 1987; Williams, 1989; Offer, 1990;
Martin and Nisbet, 1992). Recent work with different
strains of yeast and respiration-deficient yeast mu-
tants demonstrates that the ability of yeast to
stimulate the viable count in the rumen depends on its
respiratory activity (Newbold et al., 1993).
It
is
proposed that yeast removes some of the
0 2
that
occurs in ruminal fluid at various times during the
RUMINAL MICROBIOLOGY AND BIOTECHNOLOGY
2995
daily feed cycle (Hillman et al., 1985) and, therefore,
prevents toxicity
t o
the ruminal anaerobes. Less
attention has been focused on the precise mode of
action
of
A.
oryzae,
but again, the activity is destroyed
by autoclaving but not by irradiation (Newbold et al.,
1991). Thus, either a metabolic activity
or
a
heat-
labile nutrient must be responsible for the stimulatory
activity.
A.
oryzae
extract differs from yeast culture in
that the viable count of fungal cells is low (Newbold et
al., 1991).
Regardless of the efficacy or mode
of
action
of
microbial feed additives, they are already in wide-
spread use. They may, in addition, offer new opportu-
nities for manipulation. Dawson
(
1992) has isolated
strains of yeast that stimulate the growth of specific
types of bacteria, thus leading the development of
additives suitable for different dietary circumstances.
It
is a relatively small step from there to introduce
new activities into yeast that have specific and novel
models of action, as will be described below.
Introduction
of
Genetically Modified
Microorganisms in the Rumen
Requirements.
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 material
into ruminal species and ensure that it is expressed,
the second is to select
a
gene product or products that
will benefit the nutrition
of
the host animal, and the
third is
to
establish a means by which the new
organism can survive. Aside from these aspects are
ethical considerations and regulatory constraints,
which are complex and changing. They will not be
discussed here. Researchers must
first
establish
whether the objectives are achievable.
Cloning
of
Genes fvom Ruminal Microorganisms.
Since the first report
of
the successful cloning
of
cellulases from
Fibrobacter succinogenes
into
E. coli
by
Crosby et al. (1984), there have been many reports
of
genes being cloned from ruminal microorganisms into
other expression systems. Hespell
(
1989) summarized
the studies up to that time. Many more papers have
been published since 1990; these are listed in Table 1,
along with the genes cloned and the vector systems
used.
Several patterns become clear from examination
of
Hespell (1989) and Table 1: 1) The vectors and
expression are predominantly based on common
E.
coli
systems, such
as
pUC-related plasmids,
h
phage,
or
cosmids. Notable exceptions are the experiments by
Whitehead and Hespell (1990) and Whitehead et al.
(
19911, who used
Bacteroides fragilis, B. uniformis,
and
B. thetaiotaomicron
expression systems.
A
Bac-
teroides
cloning system might be expected
t o
be
superior
t o
the
E. coli
ones for cloning genes from
ruminal bacteria. The
Bacteroides
are more closely
related
to
ruminal organisms than
E. coli
and
translational and post-translational factors would be
expected to be more similar. 2) Genes have been
cloned from many
of
the predominant species of
ruminal bacteria, but only recently from the ruminal
anaerobic fungi, by Gilbert et al. (1992) and Xue et
al. (1992a,b). Because the fungi are eukaryotes,
cloning must be done via the isolation of mRNA and
preparation
of
cDNA. To date, no genes have been
cloned from the other ruminal eukaryotes, the ciliate
protozoa. 3) The enzymes that have been cloned have
been with a single exception exclusively glycosidases
or polysaccharidases, particularly xylanases and en-
doglucanases. In part this reflects the importance that
is attached to fiber digestion in the rumen, but there
are also other reasons. The cloned genes can be readily
detected using chromogenic substrates, and the en-
zymes are relatively stable. In addition, advances are
being made rapidly in this area in the field of
nonruminant research, with the result that many
genes and sequences are available for comparative
purposes, enabling fundamental aspects of the origins
and evolution of the genes to be analyzed.
Genetic and sequence studies have revealed that
some polysaccharidase genes have a simple structure
(Hazlewood et al., 1990; Cavicchioli et al., 1991; Utt
et al., 19911, and other have several domains with
different functions. Several
of
the endoglucanases and
xylanases have multiple catalytic domains of the same
(Gilbert et al., 1992; Xue et al., 1992a)
or
different
catalytic properties (Zhang and Flint, 1992; Flint et
al., 1993). Other domains are known
t o
be involved in
binding to the substrate (McGavin and Forsberg,
1989), whereas yet others are sequences
of
unusual
amino acid composition that may form hinges in the
gene product (Gilbert et al., 1992; Zhang and Flint,
1992; Flint et al., 1993). The functions of other
sequences are as yet unknown (Lin and Thomson,
199
la).
Sequence homologies also establish the ances-
tral origins
of
the different genes (Gilbert et al., 1992)
and the scope of the different enzyme families that are
involved in fiber breakdown (Ohmiya et al., 1989;
Berger et al., 1990; Lin et al., 1990; Mannarelli et al.,
1990; Matsushita et al., 1990; Cavicchioli et al., 1991;
Cunningham et al., 1991; Lin and Thomson, 1991b;
Utt et al., 1991; Whitehead and Lee, 1991; Gilbert et
al., 1992; Vercoe and Gregg, 1992; Wang and Thom-
son, 1992; Flint et al., 1993).
These studies given fascinating insight into the
variety of enzyme activities necessary to digest plant
fiber. The complex nature
of
the polypeptides
presumably reflects adaptations that have taken place
to cope with the difficulties
of
hydrolyzing insoluble
and sometimes anhydrous substrates. The work
is
particularly useful when used in combination with
enzymological studies in which cellulases and
xylanases of ruminal bacteria seem
t o
be highly
heterogeneous (e.g., Lin and Thomson, 1991b).
Genetic characterization generally indicates that there
2996
WALLACE
E
&
0
m
a,
M
cu
0
RUMINAL MICROBIOLOGY
are fewer genes than bands on zymograms, suggesting
that the heterogeneity observed in the zymograms is
due in 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 rumen
has not yet led
t o
genetic work other than the cloning
of glutamine synthetase from
B. fibrisoluens
and its
characterization as a type I11 enzyme typical
of
gut
Bacteroides
(Goodman and Woods, 1993).
It
must be emphasized that cloning genes from
ruminal organisms is extremely worthwhile, leading
to an understanding
of
the genes, enzymes, and
genetic regulation in ruminal organisms. Such fun-
damental information is essential for progress to be
made. Cloning and characterizing genes from ruminal
microorganisms does not lead directly to the objective
of using recombinant techniques
t o
improve ruminal
fermentation, however.
Expression
of
Foreign
DNA
in Ruminal Bacteria.
Construction of an improved, genetically engineered
ruminal microorganism will depend on developing
suitable genetic systems for the introduction of new
DNA into ruminal species. Progress in this area is
summarized in Table 2.
Teather (1985) reported the first instance in which
foreign DNA was transferred into
a ruminal microbial
species. The promiscuous plasmid RP4 was introduced
into
Butyrivibrio fibrisoluens
by conjugal transfer from
E. coli,
the successful transfer being demonstrated by
the 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 et al. (1988). They proved
that the antibiotic resistant was plasmid-mediated by
showing that all resistant cells had acquired a
90.5-kbp plasmid pRRI4. Russell and Wilson (1988)
used the
B. fragilis
R751 plasmid containing a pE5-2
shuttle vector to transfer erythromycin resistance
AND
BIOTECHNOLOGY
2997
between
E. coli
and
P.
ruminicola.
Hespell (1989)
reported brief details of filter matings between
Strep-
tococcus faecalis
and
B. fibrisohens
in which the
plasmid 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 l a). Tetracycline resistance was transferred
by filter-mating
Enterococcus faecalis
with
B. fibrisol-
uens.
The chromosomal insert Tn916
of
E. faecalis
was
shown
to
insert at one
or
more separate chromosomal
sites for four different
B. fibrisolvens
strains, each
representing 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 the
transposon Tn196 were conjugated from
E. faecalis
to
Streptococcus bovis.
The
S.
bovis
transconjugants were
then used as donors for matings with
B. fibrisoluens.
Successful transfer was obtained with both vectors,
although the frequency of transfer of Tn916 was very
low
at
10-7
or less.
Electrotransformation,
or
electroporation, whereby
cells that are subjected to a high electromagnetic field
become susceptible to transformation, has made the
transfer of foreign genes into ruminal microorganisms
a great deal easier, particularly with larger plasmids.
Thomson and Flint (1989)
first
showed the effective-
ness of this procedure
for
P. ruminicola,
again with
the plasmid pRRI4. The method
of
choice in construct-
ing vectors
for
use either in conjugal transfer
or
electroporation is now to make chimeric shuttle
vectors from well-characterized
E. coli
or
Bacteroides
plasmids and endogenous plasmids
of
ruminal species.
Such vectors have been effective in transfers between
E. coli
and
P.
ruminicola
as
well as different
Bacteroides
species (Shoemaker et al., 1991; Thomson
et al., 1992).
A
cryptic plasmid (pBfl ) from
B.
Table 2. Transfer and expression
of
genes in ruminal bacteria
Authors Gene Rumen species Method
Flint 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 resistance
Erythromycin resistance
Tetracycline resistance
Tetracycline, erythromycin resistance
Tetracycline, erythromycin resistance
Tetracycline, erythromycin resistance
Erythromycin resistance
Erythromycin, clindamycin resistance
Ampicillin resistance
Chloramphenicol resistance
Erythromycin resistance
Multiple antibiotic resistance
P.
ruminicola
P,
ruminicola
P.
ruminicola
B.
fibrisoluens
S.
bouis
P.
ruminicola
R.
albus
P.
ruminicola
B.
fibrisoluens
B.
fibrisolvens
S. bouis
P.
ruminicola
Conjugation with broad host range
Conjugal transfer, shuttle vector
Electrotransformation with plasmid
Conjugation with transposon
Conjugation with transposon
Shuttle vector
Electrotransformation with plasmid
Electrotransformation, conjugation
Electrotransformation, shuttle vector
Electrotransformation, shuttle vector
Electrotransformation, shuttle vector
Conjugation, electrotransformation
plasmid
with shuttle vector
with shuttle vector
2998 WALLACE
fibrisolvens
was combined with an
E. coli
pUC18
plasmid and
a
B. frugilis
clindamycin resistance gene
to give a shuttle vector that can be used to transform
both
E.
coli
and
B.
fibrisolvens
(Ware et al., 1992).
The
first
successful transformation of a
Ruminococcus
species was achieved using a lactococcal vector pCK17
and pSC22, which were electroporated into
R.
albus
by
Cocconcelli et al. (1992).
Phages offer different opportunities as vectors.
Large DNA fragments may be cloned, and, if the
phage is lytic, problems associated with the detection
of cloned genes and intact bacteria are overcome when
the bacteria are lysed during the natural course of
phage infection and replication. Many different mor-
photypes
of
phages are present in ruminal fluid
(Klieve and Bauchop, 1991).
A
temperate phage was
isolated from
S.
ruminantium
(Lockington et al.,
1988), whereas lytic phages were found in
P. rumini-
cola
(Klieve et al., 1991) and
S.
bouis (Klieve and
Bauchop, 1991). However, the use
of
these phages as
vectors has not been described.
So
far, only evidence
of
gene transfer has been
discussed.
To
make genetically engineered ruminal
microorganisms useful, the new genes will have to be
integrated into the chromosome, as was done with
B.
thetaiotamicron
(Whitehead et al., 1991); they will
have to be transcribed efficiently; they may need to be
altered by post-transcriptional modifications; and
especially in the case of polysaccharidases they may
need
t o
be secreted from the cell. Some
of
these
enzymes have signal peptide sequences (Hazlewood et
al., 1990; Gilbert et al., 1992; Flint et al., 19931, but
others do not (Mannarelli et al., 1990; Wang and
Thomson, 1990; Cunningham et al., 1991; Lin and
Thomson, 1991a; Utt et al., 19911, and a cytoplasmic
or periplasmic location
of
the gene product has been
common in
E. coli
(Berger et al., 1990; Cavicchioli and
Watson, 1991; Hu et al., 1991). The choice of species
t o
carry the new property may also be made more
difficult by restriction endonucleases, which make
introduction
of
foreign DNA into some species more
difficult than others (Lee et al., 1992; Morrison et al.,
1992a,b; Vanat et al., 1993). Clearly much develop-
ment remains
t o
be done, but the prospects for
achieving the objective of producing genetically en-
gineered ruminal microorganisms are excellent. In
principle, there seems no reason, given time and
effort, that genetic systems cannot be developed for
ruminal bacteria that can be as effective as those
developed for the main expression species such as
E.
coli.
Potentially Useful
New
Properties.
Most of the
reviews mentioned above (Smith and Hespell, 1983;
Teather, 1985; Forsberg et al., 1986; Hespell, 1987,
1989; Russell and Wilson, 1988) made comprehensive
suggestions of the new properties that might be
introduced into ruminal microorganisms and benefit
ruminant nutrition. Although virtually all the molecu-
lar work has dealt with cellulases and xylanases,
there is an evolutionary argument that questions
whether the introduction
of
microbes with different
cellulases will improve fiber breakdown. One might
expect that if a new cellulase were
t o
be beneficial, it
would already have evolved; the more optimistic view
is that evolution
of
cellulolytic ruminal microorgan-
isms is not complete. Whichever is true, there
is
certainly one instance, as pointed out by Russell and
Wilson (1988), in which cellulases could be inserted
into ruminal bacteria and be expected to be beneficial.
If cellulases were produced by organisms that are
more tolerant
t o
lower ruminal pH than the existing
cellulolytic organisms, fiber digestion might be ex-
pected
t o
improve in animals receiving a high-
concentrate ration. Even then, from the knowledge
that we already have about the complexity of the
polypeptides involved in fiber breakdown, it seems
likely that several genes will be required. The
evolutionary argument may be wrong, however, and
perhaps the single gene would markedly improve
cellulolysis. It is important
to
find out by doing the
appropriate experiments.
Ruminal protein metabolism
is
arguably a more
wasteful process nutritionally, and one that should be
approached by genetic techniques. Proteins, peptides,
and amino acids are broken down in the rumen,
destroying much of their nutritive value (Wallace and
Cotta, 1988). Many species and enzymes are involved
in proteolysis (Wallace and Cotta, 1988), but
P.
ruminicola
seems to be predominant in peptide
breakdown (Wallace and McKain, 1991; McKain et
al., 1992). Ionophores are partly effective in slowing
ammonia production, probably by
a
combination
of
removing some key species
of
amino acid-fermenting
bacteria (Russell et al., 1991) and altering the
peptidolytic and deaminative activity
of
others (New-
bold et al., 1990). Nevertheless, no direct inhibitors
of
protein
or
peptide hydrolysis have yet been discovered.
Ciliate protozoa are extremely active in engulfing and
digesting ruminal bacteria, and thus decrease the net
growth yield in the rumen (Demeyer and Van Nevel,
1979). Antiprotozoal factors would, therefore, also be
useful properties
t o
introduce
t o
improve protein
nutrition
of
ruminants.
Detoxification
is
another area that could be very
fertile for genetic manipulation. Gregg and Sharpe
(1991) described the background and problems as-
sociated with introducing bacteria that metabolize
fluoroacetate, a common toxic component
of
Aus-
tralian plants. Given the number and variety of toxic
plant components that exist (Singleton and Kratzer,
1969; Dawson and Allison, 1988), the potential here
t o
improve nutrition by genetic means must be high.
Alternatively, the new organism might be used as a
packaging mechanism for delivering enzymes or small
molecules
t o
the lower gut. Improving the amino acid
profile of microbial protein
t o
increase, for example,
RUMINAL MICROBIOLOGY
AND
BIOTECHNOLOGY
2999
lysine and methionine content was discussed by
Teather (1985) and remains a viable objective.
Establishment
of
New
Organisms
in
the
Rumen.
The best example of the successful introduction of a
new (but not genetically modified) organism in the
rumen was the introduction
of
bacteria that degraded
3-hydroxy-4(1H)-pyridone
(
DHP)
into Australian
ruminants. These animals were unable to use
Leu-
caena leucocephala
because the non-protein amino
acid, mimosine, was converted to DHP, but no further.
The DHP then had goitrogenic effects in the host.
However, when the animals were inoculated with
ruminal fluid from goats adapted
t o
consume
L.
leucocephala,
the DHP was further metabolized, thus
avoiding the toxicity
of
effects of DHP (Jones and
Megarrity, 1986; Dawson and Allison, 1988). Also of
interest are reports that certain species
of
ruminal
organism can be used
t o
inoculate the rumen
t o
enhance rumen function.
For
example, when
Megasphaera elsdenii
was added intraruminally, a
large inoculum decreased the severity
of
acute acidosis
in cattle and smaller doses enhanced recovery
of
ruminal pH (Robinson et al., 1993). Inoculation of
lambs with cultures
of
the rumen fungus,
Neocal-
Zimastix frontalis,
promoted rumen function and lead
t o
earlier weaning (Theodorou et al., 1990).
The prospective new property
t o
be introduced into
a modified ruminal organism may sometimes select in
favor of the modified strain. The prospects for
maintaining such
a
strain would be good.
For
exam-
ple, the organism described above carrying cellulase
activity and that was able
t o
grow at pH lower than
indigenous cellulolytic bacteria would actually have a
selective advantage under conditions in which
it
would
be most useful (Russell and Wilson, 1988), namely
when a mixed diet is fed, the pH falls below
6.0,
and
cellulolysis
is
compromised. Similarly, new strains
that detoxify components
of
the diet, such as the DHP
degraders mentioned above, may be competitive,
particularly if they gain energy from the metabolic
transformation. However, many inoculations, particu-
larly
of
some of the suggested types of modified
ruminal bacteria, would be
of
organisms that would
at
best be neutral with respect to selectivity (Russell and
Wilson, 1988; Gilbert and Hazlewood, 1991; Gregg and
Sharpe, 1991). The evolutionary argument comes into
play again: if
a modified organism were
t o
benefit both
itself and its host, it would already have evolved;
therefore, because it has not,
it
would be difficult
t o
establish. Furthermore, genetic modifications may
themselves impose a metabolic burden on the host
organism, making it less competitive than the wild
type. Russell and Wilson (1988) demonstrated that
the growth rate
of
a recombinant
P. ruminicola
carrying an erythromycin resistance plasmid was one-
third lower than that of the wild type, a property that
caused the resistance to be lost if the cells were
transferred more than three times in batch cultures
lacking the antibiotic.
Results obtained by tracking individual strains of
bacteria in vivo, when no external selective pressure
was applied, have been mixed. Adams et al. (1966)
found that the number
of
nonruminal bacteria added
t o
the rumen declined rapidly in vivo. A DNA probe
was 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 but
was lost in vivo at a rate corresponding to a half-life of
less than 30 min. It was suggested that the rapid
loss
was due to a bacteriocin-like activity present in
ruminal fluid (Attwood et al., 1988). Flint et al.
(1989) also found that a rifampicin-resistant strain of
Bacteroides multiacidus
was lost rapidly (49%/h),
such that the population
of
less than
lo3
mL-l was
detectable 100 h after inoculation. In contrast, a r i p
S.
ruminantiurn,
strain F100, persisted in the rumen
at
approximately
l o6
mL-l for at least
30
d. This is
not a universal property
of
S.
ruminantium,
however,
because another strain (SS2/R5) was effectively lost
within 24 h
of
inoculation, despite the antibiotic
resistance mutation in SS2/R5 causing only
a
10%
decrease in growth rate (Wallace and Walker, 1993).
Finally, Hespell (1989) cites work by
S.
D.
Mathieson,
C.
G. Orpin, and
A.
Blix in which a strain of
B.
fibrisolvens
isolated from
a
reindeer was established
successfully in a sheep at approximately
lo5
cells
mL-l. Clearly, such a small population as those
observed by Flint et
al.
(1989) and Mathieson et
al.
would be unlikely to make a significant impact on
ruminal fermentation, unless the gene product had a
secondary effect on other species. It does seem,
however, that there are factors governing the survival
of bacteria in ruminal fluid that we do not understand.
The presence
of
bacteriophages, mycoplasmas, and
bacteriocin-like toxins is well known (Hoogenraad et
al., 1967; Jarvis, 1968; Orpin and Munn, 1974; Klieve
and Bauchop, 1988). What
is
much less clear is the
effect these have on the survivability of different
strains and on the metabolic inefficiency this may
cause in ruminal fermentation.
It may be possible
t o
devise a selection strategy
whereby an ecological niche is effectively created for
the new organism in vivo
by
adding certain materials
to the feed. Many sugars and sugar alcohols that can
be utilized by nonruminant species are degraded only
slowly in rumen contents (Wallace, 1989). Experi-
ments in which sorbitol-utilizing
E. coli
or
S.
ruminan-
tium
were inoculated into the rumen and sorbitol was
added
t o
the feed as a selective energy source
for
the
growth
of
these strains were unsuccessful. The
E. coli
strain was simply unsuited
t o
growth under ruminal
conditions (Wallace et al., 19891, but the reason
for
the failure of the
S.
ruminantium
strain
t o
survive
was, as before, unknown (Wallace and Walker, 1993).
A
further problem
of
using indigenous ruminal
bacteria as vehicles
for
manipulating ruminal fermen-
3000
WALLACE
tation is their obligately anaerobic physiology. Growth
and inoculation with a suitable culture, while
straightforward 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 associated
with introducing and establishing modified ruminal
organisms that will
grow
and produce new gene
products in vivo. Indeed, given the complexity of the
problems, solutions may be difficult
t o
find.
Alternatives to Ruminal Microorganisms
for
Implementing Molecular Techniques
Other than
E.
coli, the genetics of the yeast,
5'.
cereuisiae,
are among the best known
of
all biological
organisms (Johnston,
1988).
Thus, any desired
genetic modifications could be made more readily with
S.
cereuisiae
than with ruminal microorganisms. Yeast
is already used as a feed additive, as described above,
so growth of the organism in vivo is not an issue. Live
cells are supplied at each meal and although yeast
cells grow only slowly, if at all, in the rumen, they
remain viable and metabolically active (Wallace and
Newbold,
199
2). The recombinant productfactivity
would then complement a pre-existing beneficial
action. It is logxal, therefore,
t o
suggest that, at least
in the short term,
S.
cereuisiae
might be
a
more
appropriate vehicle than indigenous ruminal organ-
isms for implementing the benefits of recombinant
DNA technology in the area of ruminal fermentation
and ruminant nutrition.
The release
of
recombinant organisms is the last
area where problems would undoubtedly be encoun-
tered, whether the organism were a ruminal species
or
S.
cereuisiae.
It is difficult to forecast how regulatory
authorities will view the use of a live recombinant
microorganism entering the food chain in ruminants.
An
even more pragmatic approach might therefore be
t o
investigate, where the desired effect can be
provided by single gene products such
as
an enzyme,
whether it may be possible to manufacture the
recombinant enzyme in a fermenter and feed directly a
purified extract containing the enzyme. The success
of
such an approach would depend on the stability
of
the
enzyme to breakdown by ruminal microorganisms,
which might be difficult
t o
achieve. It would also only
be applicable where extracellular enzymes, probably
hydrolases, rather than intracellular enzymes were
involved. However, the approach would have the
advantage that it would not require the release of a
recombinant microorganism. Dietary enzymes have
been used extensively in nonruminants (Chesson,
19931,
and indications are favorable that similar
preparations can be effective in ruminants (Konno et
al.,
1993;
Dawson,
1993).
Implications
Molecular techniques must continue
t o
be used
t o
determine the fundamental details of how ruminal
microorganisms and their enzymes carry out digestion
and fermentation. The benefits
of
this knowledge can
only be implemented, however, when other objectives,
including the development
of
genetic systems in
ruminal microorganisms and the establishment of
these modified organisms in vivo, have been resolved,
and it may be advantageous in the short term
t o
concentrate on using yeast
or
recombinant enzymes
alone
t o
produce nutritional benefits. 'When genetic
and establishment problems have been solved with
indigenous ruminal bacteria, then the added advan-
tages that they might confer, such as suitability in
extensive systems, requiring single inoculations
rather than daily feeding, will be able
t o
be exploited.
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