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Journal of Bacteriology, November 1998, p. 5921-5927, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Adherence of the Gram-Positive Bacterium
Ruminococcus albus to Cellulose and Identification of a
Novel Form of Cellulose-Binding Protein Which Belongs to the Pil Family
of Proteins
Randall S.
Pegden,1
Marilynn A.
Larson,1
Richard J.
Grant,1 and
Mark
Morrison1,2,*
Department of Animal
Sciences1 and
School of Biological
Sciences,2 University of Nebraska, Lincoln,
Nebraska 68583-0908
Received 15 June 1998/Accepted 10 September 1998
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ABSTRACT |
The adherence of Ruminococcus albus 8 to crystalline
cellulose was studied, and an affinity-based assay was also used to
identify candidate cellulose-binding protein(s). Bacterial adherence in cellulose-binding assays was significantly increased by the inclusion of either ruminal fluid or micromolar concentrations of both
phenylacetic and phenylpropionic acids in the growth medium, and the
addition of carboxymethylcellulose (CMC) to assays decreased the
adherence of the bacterium to cellulose. A cellulose-binding protein
with an estimated molecular mass following sodium dodecyl
sulfate-polyacrylamide gel electrophoresis of ~21 kDa, designated
CbpC, was present in both cellobiose- and cellulose-grown cultures, and
the relative abundance of this protein increased in response to growth
on cellulose. Addition of 0.1% (wt/vol) CMC to the binding assays had
an inhibitory effect on CbpC binding to cellulose, consistent with the
notion that CbpC plays a role in bacterial attachment to cellulose. The nucleotide sequence of the cbpC gene was determined by a
combination of reverse genetics and genomic walking procedures. The
cbpC gene encodes a protein of 169 amino acids with a
calculated molecular mass of 17,655 Da. The amino-terminal third of the
CbpC protein possesses the motif characteristic of the Pil family of
proteins, which are most commonly involved with the formation of type 4 fimbriae and other surface-associated protein complexes in
gram-negative, pathogenic bacteria. The remainder of the predicted CbpC
sequence was found to have significant identity with 72- and
75-amino-acid motifs tandemly repeated in the 190-kDa surface antigen
protein of Rickettsia spp., as well as one of the major
capsid glycoproteins of the Chlorella virus PBCV-1.
Northern blot analysis showed that phenylpropionic acid and ruminal
fluid increase cbpC mRNA abundance in cellobiose-grown
cells. These results suggest that CbpC is a novel cellulose-binding
protein that may be involved in adherence of R. albus to
substrate and extends understanding of the distribution of the Pil
family of proteins in gram-positive bacteria.
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INTRODUCTION |
The cellulosome paradigm, developed
largely from the study of Clostridium spp., is the most
firmly established example of a stable, multienzyme complex specialized
in the adherence to and degradation of crystalline cellulose (8,
12). Although high-molecular-mass cellulase complexes have also
been identified in a variety of other anaerobic bacteria, including
Bacteroides cellulosolvens, Fibrobacter
succinogenes, and Ruminococcus albus (7,
18), it is still unclear to what extent the
Clostridium spp. paradigm can be applied to these other
bacteria (7). With specific reference to R. albus, micromolar concentrations of phenylacetic acid (PAA) and
phenylpropionic acid (PPA) stimulate cellulase enzyme production
(35) and cellulose digestion kinetics (26). The
cell morphology of R. albus is also altered in response to micromolar concentrations of PAA and PPA, both vesicular and fimbrial structures are produced, and cellulases remain associated with the
bacterial capsule (34, 35). Recent analysis of the
endA gene from Ruminococcus flavefaciens revealed
a distant relationship between regions of an 80-amino-acid sequence in
EndA and the duplicated 23-amino-acid dockerin sequences found in
Clostridium thermocellum cellulosomal enzymes
(17). Even though such findings are consistent with the
hypothesis that cellulosome-like structures are produced by
Ruminococcus spp., the endoglucanase genes cloned to date
lack other features characteristic of cellulosomal enzymes. In
particular, no consensus cellulose-binding domains appear to have been
identified and characterized (15, 36), although such domains
are common in both cellulases and xylanases (14, 24).
Therefore, the mechanisms underpinning the adherence of R. albus cells to substrate, as well as cellulase secretion and
anchoring to the cell surface (and cellulose), have yet to be
elucidated. We report here the identification of two low-molecular-mass
cellulose-binding polypeptides from R. albus 8, one of which
(CbpC) possesses structural motifs typical of the Pil protein family,
which is comprised largely of type 4 fimbrial proteins produced by
gram-negative pathogenic bacteria.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
R.
albus 8 was provided by B. A. White, University of Illinois,
Urbana-Champaign; R. albus SY3 was provided by J. Miron,
Volcani Research Institute, Bet Dagan, Israel. R. albus type
strain 7 and the noncellulolytic strain B199, as well as R. flavefaciens FD-1 and type strain C-94, were obtained from M. A. Cotta, National Center for Agricultural Utilization Research, U.S.
Department of Agriculture, Peoria, Ill. The cellobiose and cellulose
(Sigmacell-19) used in growth media were obtained from Sigma Chemical
Co., St. Louis, Mo. All bacterial strains were routinely cultured in
EM-cellobiose medium (10). When necessary, strains were
grown in a defined minimal medium (34) containing either
0.2% (wt/vol) cellobiose or cellulose and transferred at least three
times prior to analysis. For cellulose-binding assays, the bacterium
was cultivated in cellobiose-containing minimal medium, supplemented
with either 5% (vol/vol) clarified ruminal fluid, 25 µM each PPA and
PAA (Sigma), or no additions.
Adherence assay.
The methods used were similar to those
described by Bayer et al. (6). Microcrystalline cellulose
(Avicel type PH-101, lot no. 1647; FMC Corporation, Philadelphia, Pa.)
was used as the substrate in all assays.
Cell fractionation procedures.
Cultures (500 ml) of R. albus 8 were grown in either EM-cellobiose or EM-cellulose medium;
following overnight (EM-cellobiose) or 72-h (EM-cellulose) growth, the
cells were harvested by low-speed centrifugation (10,000 × g, 10 min, 4°C) and washed twice with 50 mM phosphate
buffer (pH 6.5)-200 mM NaCl (phosphate-buffered saline [PBS]). The
washed cells were then resuspended in PBS prepared to contain 1 mM
phenylmethylsulfonyl fluoride and broken by two passages through a
French pressure cell (SLM Aminco Instruments, Urbana-Champaign, Ill.)
set at 250,000 kPa. Unbroken cells and large debris were removed by
low-speed centrifugation, and the membrane fragments were recovered by
ultracentrifugation (250,000 × g, 60 min, 4°C). The
supernatant was assumed to contain only cytoplasmic proteins and was
stored frozen at 
20°C until further analysis. The pellet was
resuspended in PBS containing 0.02% (vol/vol) Triton X-100, incubated
at 37°C for 15 min, and then subjected to low-speed centrifugation.
The supernatant fraction was retained and assumed to contain
solubilized membrane proteins. The sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profile
of the solubilized membrane proteins was indistinguishable from that of
crude membrane fragments (data not shown), suggesting the
solubilization procedure provided a representative sample of the
membrane proteins produced by this bacterium.
Cell-free culture fluids were concentrated by ultrafiltration in an
Amicon TCF-10 manifold fitted with a YM10 membrane
(10,000-molecular-weight cutoff). The retentate was washed and
resuspended in PBS containing 1 mM phenylmethylsulfonyl fluoride and
then stored at 20°C until further analysis.
Identification of cellulose-binding proteins by an affinity-based
assay.
Aliquots of the solubilized membrane proteins (~2 mg) or
proteins concentrated from cell-free culture fluids (100 µg) were mixed with 100 mg of Sigmacell-19 cellulose, suspended in a solution of
PBS prepared to contain 4 mM CaCl2 and 2 mM dithiothreitol, and adjusted to a final volume of 1 ml with the same buffer. This mixture was left at room temperature for 30 min with occasional agitation, and the cellulose particles were then harvested by low-speed
centrifugation. The resulting pellet was subsequently washed with
either sterilized water, PBS, various detergent solutions, or a 10%
(wt/vol) filter-sterilized solution of cellobiose. Aliquots of each
wash fraction and the cellulose particles were mixed with SDS-PAGE
running buffer, boiled, and subjected to SDS-PAGE according to standard
procedures. The stacking and resolving gels consisted of 4 and 10%
(wt/vol) T, respectively, and proteins were visualized by silver staining.
CbpC cellulose binding in the presence or absence of CMC.
Aliquots (25 µg) of the CbpC protein concentrated from culture fluids
were mixed in assay cocktails as described above, but with the amount
of cellulose present ranging from 1.6 mg to 100 mg. To assess the
effects of carboxymethylcellulose (CMC) on CbpC binding to cellulose, a
solution of CMC prepared in PBS was added to some of the mixtures prior
to the addition cellulose, to give a final concentration of 0.1%
(wt/vol). The cellulose particles were recovered by centrifugation,
washed once with PBS, and then centrifuged again. The cellulose
particles with bound proteins were resuspended in 50 µl of SDS-PAGE
running buffer and boiled, and the eluted proteins were subjected to
SDS-PAGE.
Protein sequencing and DNA techniques.
The solubilized
membrane proteins and the proteins in concentrated cell-free culture
fluids were subjected to SDS-PAGE and electroblotted onto a
polyvinylidene difluoride membrane by using a Mini Trans-Blot system
(Bio-Rad Laboratories). The membrane was stained for 2 min in a
solution of methanol-water (40:60) containing 0.005% (wt/vol)
bromophenol blue and destained with a solution of methanol-water
(50:50). The CbpC protein was cut from the membranes with a sterile
razor blade. The intact CbpC protein as well as peptide fragments
generated by trypsin digestion was subjected to Edman degradation, and
the primary amino acid sequence was determined with a Procise-HT
Microsequencer, made available through the University of Nebraska
Protein Core Facility.
Standard recombinant DNA procedures were used (
4,
33), and
enzymes were obtained from either Promega or Gibco BRL. Chromosomal
DNA
from all
R. albus strains was isolated according to standard
procedures (
4), following lysis of the cells in 10 ml of 50
mM phosphate buffer (pH 6.0) containing mutanolysin (200 U/ml),
proteinase K (150 µg/ml), and 0.5% (wt/vol) SDS for 1 h at
55°C.
The amino acid sequences obtained from CbpC amino terminus, and
tryptic fragments, were back-translated to generate oligonucleotide
primers suitable for PCR amplification of the
cbpC gene. The
PCR
mixture (50 µl) contained 20 ng of
R. albus 8 genomic
DNA, 1 µM
each primer A (5'-ATG ATG GGN TAY GTN AAG AA-3';
complementary
to the antisense strand of the sequence 24-MMGYVKK-30 of
the mature
CbpC protein) and primer B (5'-GCY TCN GGR TAY TGN CCN
AC-3';
complementary to the nucleotide sequence encoding amino acids
143-VGQYPEA-149 [see Fig.
5]), 200 µM deoxynucleoside
triphosphates,
1.5 mM MgCl
2, 50 mM KCl, 10 mM Tris-HCl (pH
9.0), 0.1% (wt/vol)
Triton X-100, and 2.5 U of
Taq
polymerase (Promega). The thermal
cycling parameters were 30 cycles for
1 min at 95°C, 1 min at
40°C, and 1 min at 72°C, followed by 1 cycle for 5 min at 72°C.
The resulting 350-bp PCR product was also
used to prepare digoxigenin-labeled
probe (Genius System; Boehringer
Mannheim) in Southern blots of
genomic DNA extracted from the
R. albus strains listed above.
Briefly,
BglII-digested DNA
was transferred to a charged-nylon
membrane (Zeta-Probe GT; Bio-Rad
Laboratories, Hercules, Calif.)
by vacuum blotting and immobilized by
UV cross-linking. The membrane
was hybridized with the probe overnight
at 65°C, washed twice
at 43°C for 30 min in 40 mM disodium
phosphate buffer (pH 7.0)
containing 1 mM EDTA and 5% (wt/vol) SDS,
and then washed once
at 43°C for 30 min in 40 mM disodium phosphate
buffer (pH 7.0)
containing 1 mM EDTA and 1% (wt/vol) SDS. The
hybridizations were
visualized according to the manufacturer's
specifications.
The nucleotide sequences of the 5' and 3' ends of the
cbpC
gene were amplified and isolated by genomic walking procedures
(Universal Genome Walker kit; Clontech). The nested primary and
secondary
cbpC gene-specific primers used for genomic
walking
procedures were based on the 350-bp sequence obtained from the
PCR product described above and were designed according to the
manufacturer's recommendations. Primary PCRs were carried out
in
25-µl volumes containing approximately 10 ng of
DraI-digested
and adapter-ligated
R. albus 8 genomic DNA, 200 µM each adapter
primer (AP1) and either a forward or
reverse gene-specific primer,
200 µM deoxynucleoside triphosphates,
1.1 mM magnesium acetate,
15 mM potassium acetate, 40 mM Tris-HCl (pH
9.3), and 1 U of r
Tth DNA polymerase XL (Perkin-Elmer). The
thermal cycling parameters
used were 7 cycles for 15 s at 94°C
and 3 min at 72°C, 37 cycles
for 15 s at 94°C and 3 min at
67°C, and then 1 cycle for 4 min
at 67°C. Secondary PCRs were
carried out in 50-µl volumes containing
1 µl of a 1:50 dilution of
the appropriate primary PCR mixture,
adapter primer AP2, and either a
forward or reverse nested gene-specific
primer. The other reaction
components and thermal cycling parameters
were the same as those used
for primary PCR. The genomic walking
was performed on two separate
occasions; independent PCR products
were sequenced by the DNA
sequencing facility at Iowa State University
and analyzed with the
Wisconsin Package (Genetics Computer Group,
Madison, Wis.).
Northern blot analysis.
Total RNA was isolated from R. albus 8 cultures grown in cellobiose minimal medium containing
either 5% (vol/vol) clarified ruminal fluid, 25 µM each PAA and PPA,
or no additions. Cells were harvested in mid-logarithmic phase of
growth (optical density at 600 nm of ~0.5) by anaerobic
centrifugation, and resuspended in 5 ml of 25 mM sodium phosphate
buffer (pH 7.0) containing 25 mM EDTA. Cells were lysed by the addition
of mutanolysin (20 U/ml), proteinase K (100 µg/ml), and 0.5%
(wt/vol) SDS and incubation at 55°C for 5 min. Total RNA was
extracted from the cell lysate by the RNeasy purification system
(Qiagen, Valencia, Calif.) according to the manufacturer's
recommendations. The RNA was concentrated by ethanol precipitation and
stored at 80°C. For Northern blots, total RNA was fractionated in
1% (wt/vol) agarose gels in the presence of both glyoxal and dimethyl
sulfoxide according to standard procedures (5, 33). The RNA
was transferred to a Zeta-Probe GT membrane by vacuum blotting and
immobilized by UV cross-linking. The cbpC-specific
radiolabeled probe was prepared by random primer labeling (Boehringer
Mannheim). Hybridization was performed overnight at 43°C, and the
blot was washed as described above for Southern blots. To determine if
there were differences in RNA loading, blots were stripped and reprobed
with a 32P-end-labeled 16-mer oligonucleotide complementary
to virtually all known 16S rRNA sequences (5'-TAC CGC GGC TGC TGG
CAC-3').
Nucleotide sequence accession number.
The nucleotide
sequence described in this article will be available in the
GenBank/EMBL databases under accession no. AF089753.
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RESULTS |
Adherence of R. albus 8 to cellulose.
Although
neither growth rate nor cell yield was affected by the addition of
either ruminal fluid or PAA-PPA to cellobiose growth medium, the
adherence of R. albus to cellulose was significantly increased by these additions. The adherence values (percentage) obtained for cells harvested during mid-log phase of growth were 65.6 ± 2.54, 73.0 ± 3.37, and 79.5 ± 3.74 for cells cultured in the
presence of no supplements, PAA-PPA and ruminal fluid, respectively. When cells were harvested during early stationary phase of growth (optical density at 600 nm of 0.8 to 1.0), the relative number of
adherent cells decreased but the effects of ruminal fluid or a
combination of PAA-PPA were maintained (data not shown). Therefore, all
subsequent assays were performed with cells cultivated in EM-cellobiose
medium and harvested during mid-log phase of growth. Similar to earlier
studies with another strain of R. albus (25), inclusion of CMC in the adherence assay mixtures decreased, but did not
eliminate, the binding of cells to cellulose. The effect of CMC was
concentration dependent, up to 0.1% (wt/vol), where adherence values
had decreased to approximately 30% (data not shown). Taken together,
the results of these experiments suggest the adherence of R. albus cells to cellulose is positively impacted by components
present in ruminal fluid such as PPA and PAA and that the cellulose
analog CMC effectively inhibits the adherence process.
Identification of two low-molecular-mass, cellulose-binding
proteins from R. albus 8 cell fractions.
When membrane
proteins extracted from cellulose-grown cells were assayed, two
polypeptides of ~21 kDa (CbpC) and ~16 kDa (CbpD) appeared to bind
completely to cellulose. Elution of both CbpC and CbpD from cellulose
particles was achieved with a solution of 0.1% (vol/vol) Triton X-100,
although 10% (wt/vol) cellobiose was also capable of removing lesser
amounts (Fig. 1). Subsequent experiments
showed that the CbpC protein could also be removed by boiling the
cellulose in SDS-PAGE loading buffer. Therefore, the limited amount of
CbpC protein present following this step in Fig. 1 (lane 8) indicates
that virtually all of the CbpC protein was removed from cellulose by
Triton X-100 and cellobiose. Further examination of the CbpC protein
showed that unlike the low-affinity cellulose-binding domains of some
clostridial proteins (reference 24, for example),
sterile water did not result in CbpC elution from cellulose (data not
shown). The results obtained with membrane proteins extracted from
cellobiose-grown cells were somewhat different from those shown in Fig.
1. The relative amount of CbpC appeared to be less, and CbpD was not
detectable. When the membrane, cytosolic, and cell-free supernatant
fractions of cellulose- and cellobiose-grown cultures of R. albus 8 were compared by SDS-PAGE analysis, a greater abundance of
CbpC was present in the membrane fractions of cellulose-grown cells,
and a polypeptide of similar molecular mass was also clearly evident in
the cell-free supernatant of the same cultures (data not shown). The
cell-free supernatant of cellulose-grown cultures was subsequently
concentrated by ultrafiltration and used in cellulose-binding assays,
and the results were virtually identical to those observed for CbpC
(Fig. 2). The protein bound tightly to
cellulose in functional assays and was largely removed by washing the
particles with 0.1% (vol/vol) Triton X-100; lesser amounts eluted with
10% (wt/vol) cellobiose. Protein sequence analysis ultimately
confirmed that the cellulose-binding proteins in both membrane and
culture fluid fractions were identical (see below), and all subsequent
assays were performed with the CbpC protein obtained from culture
fluids.
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FIG. 1.
SDS-PAGE analysis of cellulose affinity (binding) assays
using membrane proteins isolated from cellulose-grown cultures of
R. albus 8. Lanes: 1, protein molecular mass standards; 2, aliquot of solubilized membrane proteins; 3, proteins unbound after
incubation with cellulose; 4 and 5, aliquots of separate washes of the
cellulose with 10 mM phosphate buffer; 6 and 7, proteins removed from
cellulose by washing with 0.1% (vol/vol) Triton X-100 and 10%
(wt/vol) cellobiose, respectively; 8, aliquot of SDS-PAGE running
buffer mixed with the cellulose and boiled. CbpC and another
low-molecular-mass cellulose-binding polypeptide are marked with
arrows. Sizes are indicated in kilodaltons.
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FIG. 2.
SDS-PAGE analysis of cellulose affinity assay using
proteins concentrated from the cell-free supernatant of cellulose-grown
cultures of R. albus 8. Lanes: 1, protein molecular mass
standards; 2, aliquot of concentrated extracellular proteins; 3, proteins unbound following incubation with cellulose; 4 through 7, aliquots of four separate washes of the cellulose particles with 10 mM
phosphate buffer (pH 6.5); 8 and 9, proteins removed from cellulose
following treatment of the cellulose particles with 0.1% (vol/vol)
Triton X-100 and 10% (wt/vol) cellobiose, respectively; 10, proteins
removed from cellulose particles after resuspension and boiling in
SDS-PAGE running buffer. Sizes are indicated in kilodaltons.
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CbpC binding to cellulose is impaired in the presence of CMC.
In accordance with the observation that CMC blocked the adherence of
R. albus whole cells to cellulose, we chose to examine whether CMC exerted a similar effect on the binding of the CbpC protein
to cellulose. In the experiments shown in Fig.
3, the concentrations of inhibitor (CMC,
0.1% [wt/vol]) and protein (CbpC) remained constant, but the amount
of substrate (cellulose) was varied. In assay mixtures containing 25 to
100 mg of cellulose, all of the CbpC protein was bound to cellulose,
irrespective of the presence or absence of CMC (data not shown).
However, with lesser amounts of cellulose (from 1.25 to 12.5 mg), the
amount of CbpC bound was dependent on the amount of cellulose present, and in the presence of CMC, CbpC binding was further reduced, as is
clearly illustrated in Fig. 3. This is analogous to CMC exerting its
effect on the Km (of CbpC for cellulose) but not the Vmax (of CbpC bound to cellulose),
reflective of CMC serving as a competitive inhibitor of CbpC binding to
cellulose; the positional interactions of CMC and cellulose with the
CbpC protein are similar.
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FIG. 3.
Analysis of CbpC binding to increasing amounts of
cellulose, in the presence or absence of 0.1% (wt/vol) CMC. (A)
Relative amount of the CbpC protein preparation bound to either 12.5 mg
(lane 1), 6.25 mg (lane 2), 3.13 mg (lane 3), or 1.56 mg (lane 4) of
cellulose particles as described in Material and Methods; (B) results
obtained when CMC was added prior to cellulose in the assay buffer.
There were no visible differences in CbpC binding to cellulose in the
presence or absence of CMC when the amount of cellulose in the assay
was increased to 25 mg or above.
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Nucleotide and amino acid sequence analysis of CbpC.
The
33-residue amino-terminal sequence of CbpC was
XTLVELVVVIAIIGVLAAILVPSMMGYVKKARL, where X represents an
unidentified amino acid residue. When primers A and B were used
together in PCRs, a 350-bp amplification product was routinely
produced. Southern blot analyses of BglII-digested R. albus 8 genomic DNA using the 350-bp PCR product as a probe showed
that two fragments (~1.8 and ~6 kb) reacted strongly with the probe
(Fig. 4). In addition, the PCR product
cross-hybridized with genomic DNA prepared from the other R. albus strains (Fig. 4), indicating that there are regions of
homology to the cbpC gene present in these other strains. Our initial attempts at cloning and propagating the DNA fragments from
R. albus 8 proved unsuccessful, and the entire
cbpC gene sequence was ultimately obtained by a combination
of genomic walking procedures and PCR (Fig.
5). Using these methods, we identified an
open reading frame (ORF) which would encode a protein of 169 amino
acids with a calculated molecular mass of 17,655 Da. Although the
molecular mass is slightly smaller than that of CbpC estimated from
SDS-PAGE, amino acid residues 10 through 43 and 143 through 149 in the
predicted amino acid sequence were identical to the peptide sequences
obtained from purified CbpC. We therefore conclude that the DNA
sequence obtained by PCR and genomic walking procedures contains the
entire cbpC gene. Nucleotide sequence analysis also identified a BglII site within the cbpC gene,
explaining why the PCR product hybridized strongly to two fragments of
BglII-digested genomic DNA from R. albus 8.
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FIG. 4.
Southern blot analysis of genomic DNA isolated from
R. albus 8 (lane 1), 7 (lane 2), B199 (lane 3), and SY3
(lane 4), using the 350-bp PCR product of the cbpC gene as a
probe. Genomic DNA from all strains was digested with the restriction
enzyme BglII, and the migration distances of standard DNA
fragments are shown on the left.
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FIG. 5.
Nucleotide sequence of the cbpC gene from
R. albus 8. The predicted amino acid sequence is shown in
single-letter code above the coding sequence; putative 10, 35, and
ribosome-binding sites are underlined; the stop codon is indicated by
an asterisk. Sequences also determined by Edman degradation of the
purified CbpC protein are double underlined, and the presumed cleavage
site to remove the leader sequence is denoted by the inverted
triangle.
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The ORF encoded by the
cbpC gene can be subdivided into no
fewer than two distinct domains, based on BLAST analysis of the
predicted CbpC amino acid sequence with those currently in the
databases. The amino-terminal third of the protein shows significant
sequence identity with amino-terminal sequences of type 4 fimbrial
precursors from the gram-negative pathogenic bacteria
Dichelobacter (
Bacteroides)
nodosus,
Moraxella bovis,
Neisseria gonorrhoeae,
and
Pseudomonas aeruginosa (Fig.
6A). The ORF encodes an eight-amino-acid
positively charged leader sequence which terminates with a G residue.
This leader sequence is followed by an F residue and the sequence
obtained from Edman degradation of the purified CbpC protein.
In other
bacterial species expressing type 4 fimbrial proteins,
the GF dipeptide
motif serves as the site of proteolytic cleavage,
and the phenylalanine
is methylated to become the first residue
of the mature type 4 fimbrial
protein (
11). The canonical glutamate
residue at position 5 of the mature protein, found in all type
4 fimbriae and Pil homologs,
is also present in CbpC, in addition
to the highly conserved
hydrophobic sequence typical of this family
of proteins. A G residue
was also present at position 58 of the
mature protein (Fig.
5), which
has been suggested by previous
analyses of type 4 fimbrial proteins to
represent the border between
the conserved amino-terminal third and
variable carboxy-terminal
two-thirds of the protein (
11).
Beyond this G residue, the predicted
sequence was found to possess
significant homology (33% amino
acid identity) to 72 and 75 amino acid
motifs tandemly repeated
13 times within a 190-kDa outer membrane
protein of
Rickettsia rickettsii (
3) (Fig.
6B). A
similar motif is also present in
one of the large virion glycoproteins
(Vp260) from
Chlorella virus
PBCV-1, where it is also
repeated 13 times in tandem (
32). Kyte-Doolittle
plots of
the CbpC protein and the type 4 fimbriae of the bacteria
described
above are shown in Fig.
7. For the sake
of comparison,
the ComG protein of
Bacillus subtilis, which
possesses the Pil-like
amino-terminal domain but is a membrane-bound
DNA-binding protein
(
1), is also included. Despite the lack
of primary sequence
identity among these proteins, CbpC and the type 4 fimbrial proteins
all possess a reasonable degree of similarity in
terms of the
spatial distribution and length of hydrophobic and
hydrophilic
stretches of amino acids. Notably, CbpC and the fimbrial
proteins
all possess a fairly long stretch of relatively hydrophilic
residues
at the carboxy terminus, while the ComG protein does not.
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FIG. 6.
(A) Sequence alignments of the CbpC amino terminus
(Ralb) with the amino-terminal sequences from the type 4 fimbrial
proteins of M. bovis (Mbov), D. nodosus (Dnod),
N. gonorrhoeae (Ngon), and P. aeruginosa (Paer);
(B) alignment of amino acid residues 43 through 128 of CbpC with the
72- and 75-amino-acid motifs present in the surface protein antigen of
R. rickettsii (Rric72 and Rric75). Identical amino acid
residues are highlighted by a dark background; similar amino acid
residues are indicated by the shaded background.
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FIG. 7.
Kyte-Doolittle plots of the deduced amino acid sequences
for R. albus cellulose-binding protein CbpC and type 4 fimbrial proteins from M. bovis, D. nodosus,
N. gonorrhoeae, and P. aeruginosa. For the sake
of comparison, the Kyte-Doolittle plot of ComG protein from B. subtilis, a nonfimbrial Pil-like protein, is included.
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cbpC transcript abundance is increased by the inclusion
of either ruminal fluid or PAA-PPA in the growth medium.
Northern
blot analysis showed that the cbpC transcript is
approximately 700 nucleotides in length, consistent with the predicted size of the gene and the transcript being monocistronic. Additionally, cbpC transcript abundance was increased in cells cultivated
in cellobiose minimal medium supplemented with either ruminal fluid or
PPA-PAA. These findings could not be explained by differences in total
RNA loading, because the 16S rRNA-specific probe hybridized with all
preparations with similar intensities (Fig.
8). The increase in transcript abundance
correlates well with the increases seen in bacterial adherence which
were also observed following growth of R. albus 8 in medium
containing either ruminal fluid or PPA-PAA.
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|
FIG. 8.
Northern blot analysis of cbpC transcript
abundance following growth in various media. Total RNA was harvested
from cells following growth in cellobiose medium containing either
ruminal fluid (lane 1), no additions (lane 2), or both PAA and PPA. (A)
Results obtained with the cbpC-gene specific probe; (B) the
same membrane probed with an oligonucleotide complementary to 16S
rRNA.
|
|
| |
DISCUSSION |
Previous microscopic examination of the ultrastructure and
adhesion of R. albus to cellulose revealed that the cells
produce a compact mat of fibers surrounding the bacterial cell wall.
These fibrous elements also were found to project outward from the
surface by as much as 600 nm, and it was concluded that this material mediated the attachment of the bacterium to cellulose (30). A more quantitiative examination of the adherence process in selected ruminal bacteria later revealed that the adherence of R. albus to cellulose could be inhibited by CMC (25).
However, despite the identification of a role for PPA and/or PAA in
stimulating capsule production and cellulose digestion kinetics by
R. albus (26, 34, 35), no mechanistic information
about the adherence process has been obtained to date. The findings
reported here provide some of the first molecular details to explain
the observations made in these earlier studies. First, the inclusion of
either PAA-PPA or ruminal fluid in the growth medium improved the
adherence of R. albus 8 to cellulose, and these additions
were also found to increase cbpC transcript abundance.
Second, the inhibition of both R. albus whole cells and CbpC
protein adherence to cellulose in the presence of CMC further
implicates the CbpC protein in the adherence process. Third, Southern
blot analysis confirmed that a number of R. albus strains
possess genes with a high degree of sequence homology to the
cbpC gene. Preliminary Western immunoblot analysis of these
other strains with anti-CbpC antibodies identified in R. albus SY3 a protein of ~25 kDa that is also a cellulose-binding protein (27). Therefore, it seems reasonable to suggest that the CbpC protein represents a novel strategy for the adherence of
gram-positive bacteria to cellulose; the relative contribution of the
CbpC protein to the adherence process in R. albus 8 and other R. albus strains requires further examination.
A conserved protein sequence implies that the domain or motif is
involved with conserved structural-functional roles. The amino-terminal
sequence of CbpC is homologous to similarly located motifs present in a
variety of proteins from gram-negative bacteria, all of which are
involved in the assembly of protein complexes at the cell surface. Many
of these proteins are also implicated in pathogenesis, especially host
cell colonization and degradation (16). However, the
identification and characterization of Pil proteins and homologs in
gram-positive bacteria appear to be limited, having previously been
shown to exist in B. subtilis (1, 9), Streptococcus gordonii (19), and
Clostridium perfringens (16). The Pil-like
proteins in B. subtilis and S. gordonii are
involved in competence factor-dependent DNA transformation, rather than attachment to surfaces and(or) protein secretion, whereas the function of Pil homologs in C. perfringens is unclear. The
results from Southern and Western blot analyses suggest that homologs of the CbpC protein appear to exist in other strains of R. albus and that this family of proteins may be more widespread
throughout gram-positive bacteria than currently recognized.
The tandemly repeated motifs within the 190-kDa surface protein of
Rickettsia spp. are thought to have a role in
rickettsia-host interaction (2, 20, 23), possibly through
host cell recognition and attachment in a manner similar to that seen
with repetitive peptides present in parasitic eukaryotes such as
Plasmodium falciparum (22). Given the
similarities in Kyte-Doolittle plots for CbpC and the type 4 fimbriae
shown in Fig. 7, it is tempting to speculate that CbpC protein is
involved with (i) the formation of a fimbria-like structure which
possesses cellulose-binding properties and (ii) plant surface
colonization. Indeed, R. albus 8 was previously shown to
produce fimbria-like structures at the polar ends of the cell,
especially when the bacterium was cultivated in the presence of PAA and
PPA (35). In other bacteria known to produce type 4 fimbriae, a proportion of these fimbriae are also shed into the growth
medium and remain there after the cells are removed by centrifugation
(21, 28). This would explain the presence of CbpC with both
membrane and cell-free supernatant fractions of cellulose-grown
cultures. Type 4 fimbrial proteins are usually of relatively low
molecular mass (20 to 25 kDa), and it was originally hypothesized that
the hydrophobic amino-terminal motif formed the core of the fimbrial
strand, facilitating a helical stacking of the subunits (13,
21). However, beyond this hydrophobic stretch of ~50 amino
acids, the proteins show limited if any sequence similarity (11,
16), although the variable regions do possess similarities in
terms of protein chemistry. The carboxy-terminal two-thirds of many of
the type 4 fimbrial subunit proteins consist of a series of short,
fairly hydrophobic domains which have the potential to form 
-sheet
structures, around which hydrophilic sequences determining
immunoreactivity are arranged (11). It is this region of the
protein that also confers specificity in terms of binding to the cell
surface. An O-linked disaccharide has been identified in this region of
some type 4 fimbrial proteins (29) and is also thought to
contribute to immunoreactivity and surface recognition. Although we
have yet to demonstrate that CbpC is glycosylated, the presence of
sugar moieties could explain the discrepancy in molecular mass
estimated for CbpC from SDS-PAGE and predicted from cbpC
sequence analysis.
Recent monomer assembly models following protein crystallography
analysis have further resolved the structure of type 4 fimbriae (29). A right-handed helical arrangement of five monomers
still results in the amino termini forming the core of the fimbrial strand, giving rise to a waffle cone morphology. These pentameric helical structures upon stacking bury the amino termini, and only the
carboxy-terminal two-thirds is left exposed, as a smooth cylinder of
high mechanical stability. Interestingly, the secondary and tertiary
structures predicted for the repetitive motifs within the 190-kDa
R. rickettsii surface protein antigen are similar in nature
to those described above for the fimbrial subunit proteins (3). If the assembly of the CbpC protein on the surface of R. albus is similar to that described above for other type 4 fimbrial proteins (29), a complex of CbpC proteins could
also resemble the characteristics associated with a larger protein
comprised of tandemly repeated domains of the same sequence. However,
even though the motifs identified within the CbpC protein appear to be
more closely related to type 4 fimbriae, proteins with similar amino-terminal motifs that are not involved with type 4 fimbrial biosynthesis and function have been described for other bacterial species. Of these, the most notable examples include the pullulanase secretion system of Klebsiella oxytoca (31) and
cellulase secretion systems of the plant pathogens Xanthomonas
campestris and Erwinia carotovora (16, 31).
Therefore, future studies dissecting the potential roles of Pil
homologs in R. albus adherence, enzyme secretion, and
cellulase complex assembly should prove to be a valuable comparative
model of this family of proteins in gram-positive bacteria.
| |
ACKNOWLEDGMENTS |
The first two authors contributed equally to the preparation of
this report.
We thank Sanjay Reddy for conducting the adherence assays and Gautam
Sarath, University of Nebraska Protein Core Facility, for the
N-terminal sequence determinations.
This research was funded in part by the University of Nebraska
Agricultural Research Division, Soypass Royalty Funds, and USDA NRICGP
96-35206-3660 and USDA-BARD US-2783-96.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: C220 AnS, Marvel
Baker Hall, East Campus, University of Nebraska
Lincoln, NE
68583-0908. Phone: (402) 472-9382. Fax: (402) 472-6362. E-mail:
ansc802{at}unlvm.unl.edu.
Journal series no. 11973, Agricultural Research Division,
University of Nebraska.
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Top
Abstract
Introduction
