APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2005, p. 4248–4253
0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.8.4248–4253.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 8
Naturally Occurring DNA Transfer System Associated with Membrane
Vesicles in Cellulolytic Ruminococcus spp. of Ruminal Origin
Athol V. Klieve,1* Melvin T. Yokoyama,2 Robert J. Forster,3 Diane Ouwerkerk,1 Peter A. Bain,1
and Erin L. Mawhinney1
Department of Primary Industries and Fisheries, Animal Science, Yeerongpilly, Queensland, Australia1; Department of Animal
Sciences, Michigan State University, East Lansing, Michigan2; and Lethbridge Research Centre, Agriculture and
Agri-Food Canada, Lethbridge, Alberta, Canada3
Received 18 October 2004/Accepted 1 March 2005
The rumen ecosystem comprises a complex of dense microbial communities of bacteria, archaea, protozoans, fungi, and
bacteriophages (18). The fermentation effected by this complex microbiota is responsible for the conversion of plant feedstuffs to compounds that can be utilized by the animal. Hence,
the fermentations and interactions of the microbes are central
to ruminant digestion and nutrition. Of considerable interest
are the exchange of genetic material between ruminal bacteria
and the mechanisms that may enable this process to occur.
While extrachromosomal elements, such as plasmids, transposons, and bacteriophages, are well known from ruminal bacteria, few examples of genetic transfer have been documented,
and such transfers are largely inferred from the acquisition of
antibiotic resistance genes (21). In addition to the transfer of
extrachromosomal genetic elements, the presence of transformation systems, in which the chromosomal DNA of the host
bacterium is “broken up” and exported from the cell, is known
for a variety of bacterial species but has not been reported for
ruminal bacteria. Naturally occurring transformation systems
are associated with the release of DNA-containing particles
from the cell and comprise two main types, phage-like systems
based on particles with a viral appearance (4, 7, 25) and cell
wall/membrane-based structures that slough off from the cell
surface that are variously referred to as vesicles, transformasomes, or blebs (3, 8, 9, 15).
The current work elucidated a transformation system associated with membrane vesicles of the ruminal cellulolytic genus
Ruminococcus, a very important genus in the fermentation of
plant material in the rumen due to its ability to digest crystalline cellulose, the most stable plant structural polysaccharide
(2, 6).
MATERIALS AND METHODS
Bacteria and culture conditions. Ruminococcus albus AR67, Ruminococcus
flavefaciens AR6, AR45, AR69, and AR72, Butyrivibrio fibrisolvens AR5, and the
bacterial culture conditions and media used have been described previously (12).
Ruminococcus sp. isolates YE71, YE73, YE74, YE75, YE76, YE78, YE82, and
YE83 were isolated from ovine rumen contents in the current work with crystalline cellulose (Whatman no. 1 filter paper or powdered cellulose [Sigma]) as
the sole source of carbon for growth.
Cellulolytic bacteria were maintained in cellulose disk (CD) broth, which
comprised two or three disks of filter paper (Whatman no. 1) cut with a paper
hole punch in 5 ml of rumen fluid (RF)-based broth (12), in which the filter paper
was the sole source of carbon for growth. Noncellulolytic bacteria and Cel⫺
mutants of strain YE71 were maintained on RF medium with cellobiose as the
sole carbon source. Cultures were incubated at 39°C.
Concentration of subcellular particles. Cellulolytic bacteria used as sources of
subcellular particles were maintained in CD broth but were transferred to RFcellobiose broth (10 or 100 ml) and grown for 1 to 2 days, depending on the rate
of growth of the strain. Subcellular particles were harvested from the culture
supernatant, separated from bacterial cells, and concentrated by ultrafiltration
(0.45-m and 0.2-m Durapore membrane filters [Millipore Corporation, Bedford, MA]) and differential centrifugation, as previously reported for the isolation and concentration of bacteriophage particles (12).
Electron microscopy. Samples of concentrated particles were examined by
transmission electron microscopy. Negative staining was performed on butvarcoated grids with either 2% phosphotungstic acid at pH 6.5 or 3% ammonium
molybdate at pH 5.1. Samples were examined using a Philips CM10 transmission
electron microscope (Philips, Einhoven, The Netherlands).
* Corresponding author. Mailing address: Animal Research Institute, Locked Mail Bag No. 4, Moorooka, Queensland 4105, Australia.
Phone: 61 7 33629483. Fax: 61 7 33629429. E-mail: athol.klieve@dpi
.qld.gov.au.
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A genetic transformation system with similarities to those reported for gram-negative bacteria was found to
be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA
was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron
microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA
was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the
adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates.
The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb
(Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA
from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost
identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA
was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion,
suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell.
Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71
was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do
so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated
transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen.
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4249
FIG. 1. Restriction endonuclease digests of the 16S rRNA genes
from Ruminococcus sp. strain YE71 that were not cut (lane 5) or cut
with CfoI (lane 2), HaeIII (lane 6), or MspI (lane 10); rRNA genes
from R. albus AR67 that were not cut (lane 9) or cut with CfoI (lane
3), HaeIII (lane 7), or MspI (lane 11); and rRNA genes from R.
flavefaciens AR45 that were cut with CfoI (lane 4), HaeIII (lane 8), or
MspI (lane 12). Lane 1 contained the 100-bp ladder (Roche) molecular
size marker.
amplified and digested with restriction enzymes CfoI, MspI, and HaeIII for
comparison with wild-type YE71 to confirm their authenticity.
Transformation experiments. A series of experiments were performed to
determine whether membrane vesicles from YE71 (fresh or refrigerated for 7
days) and AR67 were capable of transducing Cel⫺ mutants (YE71-2 and YE7111) and B. fibrisolvens AR5 (a hemicellulolytic bacterium) to a Cel⫹ phenotype
(able to degrade crystalline cellulose). The basic experiment involved harvesting
membrane vesicles (see above) from a 100-ml RF-cellobiose broth culture of
either wild-type YE71 or AR67. The vesicle fraction was resuspended in approximately 600 l of dilution solution (23) and stored at 4°C until it was used.
Recipient Cel⫺ bacteria were grown overnight in 5 ml RF-cellobiose broth
(one preparation for each treatment) and concentrated to a volume of 200 l by
centrifugation at 1,000 ⫻ g for 10 min and resuspension in dilution solution.
Under anaerobic conditions 200 l of vesicle concentrate was added to each
sample of concentrated recipient bacteria, and the mixture was incubated at 39°C
for 2 h before it was added to 5 ml CD broth and incubated for up to 3 weeks.
The negative control treatments were (i) recipient bacteria without added vesicles and (ii) vesicles added to a concentrate from sterile RF-cellobiose medium
(treated as described above for the bacterial cultures). The positive controls were
wild-type bacteria (YE71 or AR67 depending on the experiment) in CD broth
and the Cel⫺ mutants incubated in RF-cellobiose instead of CD broth (to ensure
survival during the experimental procedure). Controls were prepared and incubated like the test organisms. CD broth media were observed for signs of
cellulose degradation (disk swelling, thinning, release of fibers, disk disintegration) over a 3-week period.
RESULTS
Ruminococcus sp. In order to expand the range of cellulolytic
ruminococci available for study and the range of R. albus
strains in particular, a number of cellulolytic bacteria were
isolated in the current work from an in vitro fermentation of
rumen contents, as previously reported for other fermentations
(13, 20). The in vitro fermentor was inoculated with ovine
rumen contents, and oaten chaff-lucerne (70:30) was used as
the food source. Isolates YE71, YE73, YE74, YE75, YE76,
YE78, YE82, and YE83 were highly cellulolytic cocci that grew
predominately in pairs. PCR amplification of the 16S rRNA
gene, digestion of the PCR product with CfoI, HaeIII, and
MspI, and comparison of DNA fragment banding patterns on
electrophoretic gels showed that all isolates were members of
the same species but were also genetically distinct from R.
albus AR67 and the R. flavefaciens strains (Fig. 1). The DNA
sequence of the 16S rRNA gene of YE71 (GenBank accession
no. AY367006) was most closely related to that of R. albus 7
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Isolation and characterization of nucleic acid. Nucleic acid was isolated and
purified from concentrated samples by using standard methods (19). The conditions used for digestion of isolated nucleic acids by DNase I and RNase A
(Roche, Mannheim, Germany) and with restriction endonucleases HindIII and
EcoRI and for digestion of PCR products with CfoI, MspI, and HaeIII were the
conditions specified by the manufacturer (Roche).
The routine electrophoretic techniques were the techniques described by Maniatis et al. (19). DNA size was determined by comparison with a HindIII digest
of phage or a 100-bp DNA size marker (Roche). Pulsed-field gel electrophoresis (PFGE) was performed as previously described (14), except that the machine
used was a contour-clamped homogeneous electric field (Bio-Rad, Hercules,
CA). The DNA size marker was a phage concatemer (Bio-Rad).
PCR amplification, sequencing, and sequence analysis of 16S rRNA genes.
The 16S rRNA genes were enzymatically amplified from genomic DNA or
subcellular particles (26) using PCR methods previously described by Ouwerkerk
and Klieve (24). One nanogram of target DNA was used, and the standard PCR
assay consisted of 30 cycles.
Sequencing was performed with an ABI Prism dye terminator cycle sequencing
Ready Reaction kit with Amplitaq DNA polymerase FS and a model 373A DNA
sequencing system (PE Applied Biosystems Inc., Foster City, CA) by following
the manufacturer’s protocols. The primers used for sequencing were 27F, 530F,
926F, 1114F, 342R, 787R, 1100R, and 1525R (16). Sequencing reactions were
performed at the Griffith University DNA Sequencing Facility (School of Biomolecular and Biomedical Science, Griffith University, Queensland, Australia).
Sequence fragments were assembled using ContigExpress from the Vector NTI
Suite 6 package (Informax, Frederick, MD) (www.informaxinc.com). The
Gapped BLAST database search program (1) at the National Centre for Biotechnology Information was used to compare sequences.
The almost full-length 16S rRNA gene sequences were aligned with similar
sequences using the ARB software package (17), and a phylogenetic tree was
reconstructed using the neighbor-joining method, the Kimura correction, and an
evaluation of 1,000 bootstrapped trees with the Phylo_win program (5).
Attachment of subcellular particles to cellulose powder. To ascertain whether
the subcellular particles containing DNA were likely to be membrane vesicles,
samples of a particle concentrate from R. albus AR67 were incubated with
cellulose powder. In cellulolytic ruminococci, cellulosomes are also associated
with membrane vesicles, which attach to cellulose (10) and therefore sediment
with cellulose upon low-speed centrifugation. Whether DNA can be extracted
from cellulose-sedimented vesicles can then be determined.
The subcellular particle fraction (not concentrated) from a 100-ml overnight
culture of R. albus AR67 in RF-cellobiose broth was transferred to an anaerobic
chamber (Coy Laboratory Products Inc., Ann Arbor, MI), and 20- to 25-ml
aliquots were placed in four sterile serum bottles, two of which contained 100 mg
of cellulose powder. The serum bottles were sealed, removed from the anaerobic
chamber, and incubated at 39°C for 2 h with mixing every 15 min. Suspensions
were then transferred to centrifuge tubes and centrifuged at 300 ⫻ g for 5 min.
The pellet was retained for extraction of DNA from the cellulose fraction. The
supernatants were centrifuged again at 30,000 ⫻ g and 4°C for 2 h to pellet the
remaining subcellular particles. DNA was extracted from material pelleted in
both the low- and high-speed centrifugations.
Production of Celⴚ mutants. To demonstrate transformation, it is necessary to
have genetically distinct strains with observable phenotypes that are selectable,
such as antibiotic resistance. As libraries of genetically altered ruminococci are
not available to our knowledge, mutants of strain YE71 that were not capable of
disrupting crystalline cellulase were created.
From an overnight culture (RF-cellobiose broth), 0.1 ml of Ruminococcus sp.
strain YE71 was transferred to 5 ml RF-cellobiose broth containing 50 l of a
0.1-mg ml⫺1 ethidium bromide (Bio-Rad) or acridine orange (Sigma-Aldrich,
Castle Hill, New South Wales, Australia) solution. The cultures were incubated
overnight at 39°C prior to spread plate inoculation of 10-l portions onto either
plain RF-cellobiose agar plates or plates that had been impregnated with
ethidium bromide or acridine orange at the concentrations described above. The
plates were incubated for a further 24 to 48 h, after which well-separated colonies
were picked and duplicate plated on RF-cellobiose and RF-cellulose (powdered
cellulose) agar plates. The plates were incubated for 7 to 10 days. Colonies that
grew on RF-cellobiose but either did not grow or showed some growth but no
zone of clearing on RF-cellulose were selected for further study. Wild-type YE71
was used as a positive control. The putative Cel⫺ mutants were grown in RFcellobiose broth overnight prior to replating on RF-cellulose agar plates and
subculturing in CD broth to confirm the Cel⫺ status. The cultures were incubated for 7 to 10 days.
DNA was extracted from Cel⫺ mutants, and the 16S rRNA gene was PCR
RUMINOCOCCUS DNA TRANSFER
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FIG. 2. Phylogenetic tree indicating the relationships of Ruminococcus sp. strain YE71 and related ruminococci. Bar ⫽ 1% sequence
divergence. Database sequence accession numbers for known organisms are indicated in parentheses.
(93.2% binary similarity); however, the difference from related
ruminococci is great enough that this organism could be regarded as a separate species (24) (Fig. 2).
DNA characteristics. Nucleic acid was recovered from the
subcellular fraction of all the cellulolytic ruminococci examined. This nucleic acid was confirmed to be double-stranded
DNA as it was completely digested by DNase I but not by
RNase A. The DNA completely dissociated upon heating at
95°C. DNA profiles for selected ruminococci, as they appeared
on a conventional agarose gel, are shown in Fig. 3a. The DNA
recovered from different bacterial strains varied in appearance
on gels. Generally, one main band was present at the limit of
migration of linear DNA, just above the 23-kb DNA marker.
Some strains (e.g., the YE strains) initially produced three
bands, one at around the 23-kb marker, one faint band that
migrated more slowly, and one band that migrated much
faster, which was suggestive of plasmid DNA with supercoiled,
circular, and linear forms present. However, the profiles of
these strains tended to vary, and often just the single band at
around 23 kb was present.
PFGE of DNA from subcellular particles from R. albus
AR67 and Ruminococcus sp. strain YE73 (Fig. 3b) revealed
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that the DNA consisted of a population of linear DNA molecules that varied in length from ⬍ 20 kb to 49 kb for strain
YE73 and from 23 kb to 90 kb for strain AR67. The average
length of molecules was different in the two strains; the average
length of the YE73 molecules was approximately 30 kb, and
the average length of the AR67 molecules was approximately
50 kb. The presence of a small amount of DNA remaining in
the well may have indicated the presence of some circularized
molecules.
Restriction digestion of particle-associated DNA from R.
albus AR67 with HindIII (Fig. 4) did not result in a specific
banding profile that corresponded to the linear length of the
DNA but instead resulted in a complex profile consisting of
many bands. Digestion of genomic DNA from R. albus cells
(Fig. 4) gave a profile that was almost identical to that obtained
for the particle-associated DNA. The only discernible difference was that a couple of bands in the digest of the particle-
FIG. 4. Restriction digestion of chromosomal DNA extracted from
whole cells of R. albus AR67 (lanes 5, 6, and 7, uncut and HindIII and
EcoRI digests, respectively) and DNA from subcellular particles from
R. albus AR67 (lanes 2, 3, and 4, uncut and HindIII and EcoRI digests,
respectively). Lanes 1 and 8 contained DNA size markers. The arrows
indicate DNA bands that appear to be overrepresented in the HindIII
digest of the DNA from subcellular particles.
FIG. 5. Electron micrograph of membrane vesicle-like structures
from Ruminococcus sp. strain YE71. Bar ⫽ 50 nm.
associated DNA appeared to be overrepresented compared to
the genomic DNA digest (Fig. 4). Paradoxically, digestion of
AR67 genomic DNA with EcoRI resulted in a complex profile,
like that obtained by digestion with HindIII, but EcoRI did not
digest particle-associated DNA (Fig. 4). Furthermore, PCR
amplification of the 16S rRNA gene from particle-associated
DNA from R. albus AR67 resulted in a PCR product with a
restriction profile identical to that of the PCR product of the
16S rRNA gene amplified from AR67 genomic DNA.
Electron microscopy. No bacteriophage-like particles were
found when concentrated samples of DNA-containing particles from R. albus AR67 and Ruminococcus sp. strain YE71
were observed by electron microscopy. The only common particles that might be expected to contain DNA resembled the
membrane vesicle structures reported previously for R. albus
F-40 (10). An electron micrograph is shown in Fig. 5.
Cellulose binding. DNA was extracted from the cellulose
powder fraction following incubation with subcellular particles,
and noticeably less DNA was present in the supernatant following incubation (Fig. 6), suggesting that the subcellular particle DNA was associated with the same structures (membrane
vesicles) that were associated with the cellulosomes that had
attached to the cellulose powder.
Celⴚ mutants. Acridine orange appeared to have little effect
on the growth of Ruminococcus sp. strain YE71, and the overnight growth in the presence of this compound appeared to be
similar to the growth of control cultures. Ethidium bromide, on
the other hand, severely reduced the growth rates. Following
incubation with acridine orange and ethidium bromide, a total
of 75 colonies were selected and examined for the ability to use
powdered cellulose. Five clones that had been incubated with
ethidium bromide showed a reduced ability to grow on cellu-
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FIG. 3. DNA extracted from subcellular particles and electrophoresed on either a conventional agarose gel (a) or a PFGE gel (b). (a)
Lane 1, HindIII digest of phage DNA size marker; lanes 2 to 6, R.
albus AR67, R. flavefaciens AR69, AR72, and AR6, and Ruminococcus
sp. strain YE73, respectively. (b) Lane 1, lambda ladder size marker;
lane 2, HindIII digest of phage DNA size marker; lanes 3 and 4,
DNA from Ruminococcus sp. strain YE73 and R. albus AR67, respectively.
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KLIEVE ET AL.
lose. Two of these clones (YE71-2 and YE71-11) could not
grow on cellulose at all, and when they were inoculated into
CD broth, no signs of growth or degradation of the cellulose
disks were apparent after 3 weeks of incubation. DNA extraction, PCR amplification of the 16S rRNA gene, and restriction
digestion of the PCR product produced banding patterns identical to that of wild-type strain YE71, whose pattern was distinct from those of the other ruminococci (Fig. 1).
Transformation. Subcellular particles from wild-type strain
YE71 consistently (three trials) conferred the ability to degrade crystalline cellulose on bacterial mutants that were otherwise unable to do so. Acquisition of this property was quite
slow, and the first signs of degradation of the cellulose disks
(disk swelling, release of fibers, disk disintegration) did not
appear until 7 to 10 days, but then they continued to develop
for up to 3 weeks. CD broth media with either a mutant alone
or subcellular particles alone showed no signs of degradation
of the cellulose disks over the same period.
The acquisition of the ability to degrade crystalline cellulose
by the mutants appeared to be heritable, as cells from both
transduced mutants were sequentially subcultured twice in CD
broth and the rate and extent of cellulose degradation increased so that there was complete disintegration of the disks
within 7 days by the second subculture. The appearance of the
transformed cells was not noticeably different from the appearance of the wild type.
Transformation of the hemicellulolytic ruminal bacterium B.
fibrisolvens AR5 with YE71 vesicles was not successful.
DISCUSSION
The cellulolytic ruminococci of the rumen appear to possess
a subcellular lateral gene transfer mechanism for transformation. Double-stranded DNA was isolated from particles in culture supernatants that were filterable and sedimentable by
centrifugation. Commonly, the only nucleic acid-containing entities with these properties are bacteriophages (12), but no
particles having typical phage morphology were observed by
electron microscopy. The only “particles” that were observed
were amorphous, mainly globular material of variable size that
resembled membrane vesicles, as described by Kim et al. (10).
The presence of these particles was not surprising as the bacteria under investigation were cellulolytic ruminococci, which
are known to release cellulosome-containing vesicles. However, nucleic acid has not previously been associated with these
particles. As cellulosome-containing membrane vesicles adhere to cellulose, the addition of cellulose powder to culture
supernatant and transfer of a proportion of the extractable
DNA from the fluid to the powder suggest that the DNA is
associated with membrane vesicles.
DNA was present in culture filtrates from all of the cellulolytic ruminococci investigated, and therefore, this appears to
be a common feature in this genus. The DNA, at least the
DNA of R. albus AR67, appeared to be chromosomal DNA
from the host bacterium itself. The majority of the DNA was
linear and variable in size. The average size was different for
different species of Ruminococcus, and based on the endonuclease digestion pattern the entire genome appeared to be
present in the overall population of membrane vesicles. The
presence of the bacterial 16S rRNA gene further supports this
assertion. The amount of DNA associated with each particle
and how it is packaged remain to be determined, but it appears
that the DNA is processed and packaged for inclusion with the
particles. First, the DNA was cut into relatively short linear
pieces compared to the bacterial chromosome length. In the
HindIII digestion profile there were a couple of DNA bands
that appeared to be overrepresented in the particle-associated
DNA compared to chromosomal DNA, and these bands may
represent repetitive DNA sequences possibly used for packaging. The most intriguing aspect of the particle-associated DNA
is its refractivity to digestion with EcoRI, even though the
chromosomal DNA from the same bacterium was susceptible
to digestion with this enzyme. Restriction modification systems
are known for R. albus (22), but differential protection of DNA
exported from the bacterial cell has not been reported. In
addition to the particle-associated DNA reported here, it was
previously noted that phage DNA from R. albus AR67 was not
digested by EcoRI (11). The most plausible explanation for
this is that only DNA exiting the bacterial cell is restriction
modified, which implies that either the modification system or
the chromosomal DNA is compartmentalized. The results also
bring into question whether restriction endonucleases or just
the restriction protection system is present in the bacterial cell.
Considerably more research is required to answer these questions, but it does appear that DNA associated with membrane
vesicles is specifically processed for export from the cell.
The presence of the 16S rRNA gene has been used as an
indicator of generalized transducing phage systems in bacteria
(26). This methodology is equally relevant to other subcellular
particles that are filterable and sedimentable in a manner similar to the manner observed for phages. The particle-associated
DNA that we describe here appears to be very similar to the
“bleb-associated” or “vesicle-associated” transformation systems reported for Haemophilus influenzae (9), Neisseria gonorrhoeae (3), Pseudomonas aeruginosa (8), and Escherichia coli
(15). These gram-negative bacteria all produce surface vesicles
that slough off from the bacterial cell. The ruminococci are
gram positive, and similar systems do not appear to have been
reported previously for gram-positive bacteria. However, sim-
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FIG. 6. DNA extracted from the cellulose powder fraction following incubation with subcellular particles from R. albus AR67 (lanes 2
and 3, cellulose powder added; lanes 4 and 5, no cellulose added) and
DNA extracted from particles remaining in the supernatant following
the removal of cellulose powder (lanes 6 and 7, cellulose powder
added; lanes 8 and 9, no cellulose added). Lane 1 contained a 1-kb
DNA size marker.
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ACKNOWLEDGMENTS
We thank Andrea Turner and Lyle McMillen for technical assistance and Howard Prior for electron microscopy.
This work was funded through the Australian Research Councils’
Large Grants Scheme.
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ilar to the gram-negative bacteria, the ruminococci do produce
surface-associated vesicles that slough off from the cell (10). It
appears that the surface vesicles of the ruminococci serve a
dual purpose, degradation of plant fibrous material and lateral
gene transfer.
While the membrane vesicle system of the ruminococci has
many characteristics of a transformation system, further evidence is required to clarify the functionality. With genetically
well-defined bacterial species, such as the gram-negative
pathogens discussed above, functionality is often demonstrated
by “rescuing” genetically mutated cell lines or by transfer of
new traits, such as antibiotic resistance. Unfortunately, libraries of defined mutants of rumen bacteria in general and ruminococci in particular are not available for such studies. To
overcome this limitation, we created mutants that were unable
to grow on crystalline cellulose, which resulted in a phenotype
that was easily identifiable because of the inability to degrade
filter paper. Despite the fact that the mutants were crudely
produced and undefined, we demonstrated that concentrated
suspensions of vesicles from the wild-type bacterium were able
to consistently “rescue” these mutants and enable them to
degrade crystalline cellulose. The time necessary for the transformed mutants to show signs of cellulose utilization was long,
indicating that the frequency of transformation was probably
low, as would be expected. More importantly, the acquisition
of cellulose degradation by the mutants was heritable, as indicated by sequential subculture in CD broth media of the rescued mutants. Further work, preferably with well-defined mutants, is required to more fully understand this system.
In conclusion, it appears that the cellulolytic ruminococci
possess a transformation system associated with vesicles, giving
this organelle at least a dual role in the biology of the ruminococci. This discovery removes the limitation of such systems
to gram-negative bacteria and indicates that transformation
plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen.
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