1 Unité de Microbiologie, INRA, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France
2 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA
3 The MAPLE Research Program, Department of Animal Sciences, The Ohio State University, 2027 Coffey Road, Columbus, OH 43210, USA
Correspondence
Pascale Mosoni
pmosoni{at}clermont.inra.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the pilA2 gene sequences of R. albus strains 8 and 20 are respectively AY880680 and AJ853471.
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INTRODUCTION |
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Recent work in our laboratory has established that type IV fimbria-like structures are involved in the adhesion of R. albus 20 to cellulose (Rakotoarivonina et al., 2002). The fimbrial structural protein was first identified as a CBP called CbpC in R. albus 8 (Pegden et al., 1998
). By comparing R. albus strain 20 with its spontaneous adhesion-defective mutant (strain D5), we recently showed the presence of pili on the surface of R. albus 20 and identified a glycoprotein, initially called GP25, as the major pilus subunit (Rakotoarivonina et al., 2002
). Pil protein homologues have also been shown by Southern and Western blot analyses to be encoded in the genomes of other strains of R. albus (Pegden et al., 1998
).
In an effort to understand these adhesion structures better, we looked for the presence of genes flanking cbpC and gp25 likely to encode functions involved with the secretion and assembly of pili in R. albus. We report here the presence of a second pil gene present in both R. albus strains 20 and 8 that is located adjacent to cbpC/gp25. We examined the expression of these tandemly arranged pil genes by quantitative RT-PCR in strain 20 and mutant D5, and by Northern blot analysis in R. albus strain 8. Antibodies specific to the second pil-gene product in R. albus strain 20 were prepared and used to show that this second gene product is not part of the externalized fimbrial structure but resides mainly in the membrane of the bacterium.
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METHODS |
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Cloning and nucleotide sequence analysis of the pilA1pilA2 locus in R. albus strains 8 and 20.
Genomic DNA isolation and the cloning and sequencing of the gp25 and cbpC genes has been described previously (Rakotoarivonina et al., 2002; Pegden et al., 1998
). For both strains, genome-walking procedures were used to obtain nucleotide sequence information downstream of the genes encoding GP25 and CbpC, in R. albus strains 20 and 8, respectively (Universal Genome walker kit; Clontech). Oligonucleotide primers (Table 1
) were synthesized by either MWG Biotech or Sigma-Genosys, and the primary and secondary gp25/cbpC gene-specific primers were designed according to the manufacturer's recommendations. For R. albus 20, a PCR product encoding 1·5 kb of sequence downstream of the gp25 gene was amplified using GW1-F and the adaptor primer AP2 and PvuII-digested genomic DNA. The PCR product was purified from agarose gel slices using the Strataprep PCR purification kit (Stratagene) and sequenced by primer-walking methodology with an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Perkin Elmer).
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The DNA sequences obtained from strains 20 and 8 were analysed with the BLASTX program (Altschul et al., 1990) and the Wisconsin Genetics Computer Group (GCG) package version 9.1 to determine sequence similarities to those available in the GenBank and EMBL databases.
Isolation of the pilA2 gene from the adhesion-defective mutant D5.
To amplify a DNA fragment encoding pilA2 and its upstream region (i.e. putative promoter) in R. albus D5, a PCR was carried out with genomic DNA using primers PilA2-5'-F and PilA2-3'-R (Table 1) and 1 U AmpliTaq DNA polymerase (Appligene). The PCR parameters were an initial denaturation step (94 °C for 5 min), 25 cycles of amplification (94 °C for 90 s, 55 °C for 60 s and 72 °C for 90 s) and a final elongation step (72 °C for 7 min). The PCR product was sequenced in both directions with the same primers as those used for amplification.
RNA extraction and Northern blot analyses.
Cultures of R. albus 8 were harvested during the mid-exponential phase of growth (OD600 0·5) by anaerobic centrifugation (10 000 g, 10 min at 4 °C). The pelleted cells were washed twice with 10 ml volumes of 25 mM sodium phosphate buffer (pH 7·0) containing 25 mM EDTA. The cells were then suspended in 10 ml of the same buffer and cell lysis was accomplished by the addition of mutanolysin (200 U ml1), proteinase K (150 µg ml1) and 0·5 % (w/v) SDS for 5 min at 55 °C. The RNA was recovered from the lysis mixture by using the RNeasy purification system from Qiagen according to the manufacturer's recommendations. Total RNA was DNase-treated according to standard procedures (Sambrook et al., 1989
) and RNA integrity was checked by electrophoresis in a 1 % (w/v) agarose gel and ethidium bromide staining.
For Northern blot analyses, 10 µg total RNA for each growth condition was fractionated in 1 % (w/v) agarose gels in the presence of glyoxal and DMSO according to standard procedures (Sambrook et al., 1989). The RNA was transferred to a Zeta-Probe GT membrane (Bio-Rad Laboratories) by vacuum blotting and immobilized by UV cross-linking. The pilA2-specific probe was radiolabelled with [
-32P]dCTP by random primer labelling (Pharmacia Biotech) according to the manufacturer's recommendations and blot hybridizations were performed overnight at 43 °C. After hybridization, the membranes were washed twice at 43 °C for 30 min in 40 mM disodium phosphate buffer (pH 7·0) containing 1 mM EDTA and 5 % (w/v) SDS and then washed twice at 43 °C for 30 min in 40 mM disodium phosphate buffer (pH 7·0) containing 1 mM EDTA and 1 % (w/v) SDS. Differences in total RNA loading were determined by stripping the blots and hybridizing with a 32P-labelled oligonucleotide that is complementary to virtually all known 16S rRNA sequences (5'-TACCGCGGCTGCTGGCAC-3'; Zheng et al., 1996
). A PhosphorImager (Molecular Dynamics) was used to quantify differences in transcript abundance. Transcript size was estimated by using a standard curve produced from an RNA standard ladder (Bio-Rad) that was loaded on the same gels used for Northern analysis.
Isolation of a pilA1pilA2 cotranscript.
Amplification of a pilA1pilA2 cotranscript was carried out using cDNA obtained from R. albus 20 as template and a forward primer located in the 5' region of pilA1 (PilA1-F) and a reverse primer located in the 3' region of pilA2 (PilA2-R) (Table 1). Positive controls were also performed using either forward and reverse pilA1 primers or forward and reverse pilA2 primers. A negative control was included, using RNA not reverse-transcribed (see below) as template. PCRs were carried out with 1 U AmpliTaq DNA polymerase and 100 ng cDNA. The PCR parameters were an initial denaturation step (94 °C for 5 min), 30 cycles of amplification (94 °C for 90 s, 60 °C for 60 s and 72 °C for 90 s) and a final elongation step (72 °C for 7 min).
Analysis of gene expression using real-time RT-PCR.
Total RNA was isolated from R. albus 20 and D5 cultures grown in RF cellobiose medium. Cells (10 ml culture) were harvested in the stationary phase of growth by centrifugation and washed three times in 1 ml DEPC-treated TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8). Initial cell lysis was accomplished in 200 µl of the same buffer containing lysozyme (3 mg ml1) and proteinase K (100 µg ml1) for 1 h at 37 °C. Cells were then disrupted by bead beating for 1 min with 1 g zirconia beads. Subsequent RNA extraction was performed using the RNeasy Mini kit (Qiagen) according to the manufacturer's recommendations. The RNA eluted in 40 µl DEPC-treated water was treated with 10 U FPLC-pure DNase I (Amersham) for 30 min at 37 °C. RNA concentration was estimated by absorbance at 260 nm. RNA integrity was verified by electrophoresis of an ethidium-bromide-stained 1·4 % (w/v) agarose gel.
Total RNA (1 µg) was reverse-transcribed into cDNA using random hexamer primers (Invitrogen) and 200 U SuperscriptII Rnase H reverse transcriptase (Invitrogen) according to the procedure supplied with the enzyme. For each RNA, a negative RT reaction (no addition of reverse transcriptase) was performed to be used as a negative control in subsequent PCRs.
Real-time PCR was carried out using a LightCycler system (Roche Molecular Biochemicals). Primers for pilA1 and pilA2 yielded PCR products of 200 bp (Table 1). To control for RNA quality and cDNA synthesis, 16S rRNA was also amplified using primers that also yielded a 200 bp product. For LightCycler PCRs, a master mixture containing MgCl2 (4 mM), forward and reverse primers (0·5 µM each) and LightCycler-FastStart DNA Master SYBR Green I (2 µl per reaction; Roche Molecular Biochemicals) was prepared. Aliquots of the master mix (18 µl) were dispensed in the LightCycler glass capillaries and then various amounts of cDNA (0·1, 0·25, 0·5 or 1 ng reverse-transcribed total RNA) were added as PCR template to give a final volume of 20 µl. After denaturation at 95 °C for 10 min, the amplification and quantification program was repeated 45 times (95 °C for 15 s, 62 °C for 5 s, 72 °C for 8 s with a single fluorescence measurement), followed by the melting curve program (6595 °C with a heating rate of 0·1 °C s1 and a continuous fluorescence measurement) and finally a cooling step to 45 °C. Negative controls without cDNA template or with RNA not reverse-transcribed were run with every assay to assess the overall specificity.
Quantification involved the use of standard curves that had been prepared with PCR products corresponding to almost the entire sequence of each gene (pilA1, 520 bp; pilA2, 450 bp; rrs, 1484 bp). The pilA1 and pilA2 genes were amplified from R. albus 20 genomic DNA, using the same primers and PCR parameters as those given for the isolation of transcripts. For the rrs gene, the forward bacterial domain primer F8 (5'-AGAGTTTGATCMTGGCTC-3'; Tm 52 °C) and the reverse universal domain primer 1492R (5'-GNTACCTTGTTACGACTT-3'; Tm 52 °C) were used. For the construction of standard curves for pilA1, pilA2 and rrs genes, PCR products were prepared as a 10-fold serial dilution in water, from 102 to 108 copies. LightCycler quantification software (version 3.5) was used to compare amplification in experimental samples during the log-linear phase to the standard curve from the dilution series of control PCR products. Relative results are reported in terms of the copy number of pilA1 or pilA2 mRNA per 106 copies of 16S rRNA. Each experiment was repeated three times to confirm the reproducibility of the results.
Cloning and expression of the pilA2 gene.
The pilA2 gene was PCR-amplified using primers that removed the first 18 nucleotides encoding the signal sequence of the predicted protein and also contained NcoI and XhoI sites at their 5' and 3' ends, respectively (Table 1). The resulting PCR product was cloned into an NcoI/XhoI-digested pET28a(+) vector such that a His6 tag was fused with the C terminus of the recombinant protein. Clones were expressed in E. coli BL21(DE3) as follows: single colonies were transferred into 10 ml LB medium with 25 µg kanamycin ml1. These cultures were then used to inoculate 1 l volumes of the same medium and were allowed to grow at 37 °C with agitation to an OD600 of 0·30·5; 1 mM IPTG was then added and the culture was incubated for a further 6 h at 37 °C.
Purification of His-tagged PilA2 and preparation of anti-PilA2-His6 serum.
The PilA2-His6 recombinant protein was purified from E. coli cell extracts by NTA-resin affinity chromatography (Qiaexpressionist System; Qiagen). Briefly, cultures of recombinant E. coli were centrifuged (10 000 g, 4 °C, 15 min) and the cell pellet was resuspended in 25 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8) and incubated with lysozyme (1 mg ml1) at 4 °C for 30 min. The cell suspension was then broken by two passages through a French pressure cell set at 1250 bar (125 MPa). Unbroken cells and large debris were removed by centrifugation (10 000 g, 4 °C, 30 min) and the supernatant containing cytoplasmic proteins was loaded onto 4 ml Ni-NTA resin equilibrated with lysis buffer. Adsorbed proteins were eluted by adding 12 ml aliquots of 20, 50, 100, 150, 200 and 250 mM imidazole in lysis buffer. The PilA2-His6 recombinant protein appeared in fractions eluted with 100 and 150 mM imidazole that were pooled and dialysed against water using a regenerated cellulose membrane (Spectra/Por; Spectrum) with a molecular mass cut-off between 6 and 8 kDa. The dialysate was concentrated by centrifugation (5000 g, 4 °C, 90 min) on a Microsep concentrator with a molecular mass cut-off of 10 kDa (Pall Filtron). It was finally purified by a gel extraction procedure (Hames, 1981). The anti-PilA2-His6 serum was prepared by injecting adult New Zealand white rabbits subcutaneously with 50 µg protein (in acrylamide) that had been suspended in 1 ml sterile NaCl (0·9 % w/v) and emulsified with Freund's incomplete adjuvant. Injections were performed three times, at 3-week intervals, and the rabbits were bled 2 weeks after the last immunization. The blood was incubated without agitation for 3 h at 37 °C and centrifuged at 3000 g for 25 min. The upper, serum phase was stored at 20 °C. Blood was also collected from the rabbits prior to immunization and treated in the same manner to provide pre-immune serum.
Preparation of anti-peptides targeting PilA2.
Two peptides of 15 amino acids designated the internal peptide (MIYNSIAANAKNSG; positions 90104) or C-terminal peptide (NSVHNVPTWKAYKAD; positions 141155) were chosen to prepare specific antibodies against PilA2. These peptides were chosen to ensure that the extremely hydrophobic and highly conserved N-terminal region of type IV pilins was excluded as an epitope(s), while the C-terminal region of the protein should be quite antigenic (Hazes et al., 2000). The internal peptide was chosen by using hydrophobic cluster analysis (Gaboriaud et al., 1987
) and was predicted to reside in a loop at the boundary of
2
3 strands of type IV pilins. This sensitive two-dimensional method of sequence analysis is able to detect structural relationships between protein sequences sharing low levels of sequence identity (typically below 20 % identity).
Pre-immune serum, the antiserum obtained against the two (C-terminal and internal) PilA2 peptides hereafter referred to as anti-peptide antiserum and affinity-purified antibodies against either the C-terminal peptide (anti-PilA2-C antiserum) or the internal peptide of PilA2 (anti-PilA2-I antiserum) were prepared and provided by Eurogentec.
Western blot analysis of R. albus proteins.
After growth on RF cellobiose medium to an OD600 of approx. 1·2, R. albus 20 and the adhesion-defective mutant D5 were harvested by centrifugation (10 000 g, 4 °C, 20 min). The resulting cell pellets were washed three times in 50 mM sodium phosphate buffer (pH 6·9) and then resuspended in one-tenth of their original volume of the same buffer and frozen at 20 °C. The culture fluid, containing extracellular proteins, was also aliquotted and frozen. CBPs from the extracellular culture fluid were recovered as described previously (Rakotoarivonina et al., 2002) and membrane and cytoplasmic proteins plus cell-associated CBPs were prepared following already-described procedures (Mosoni & Gaillard-Martinie, 2001
). Western immunoblot analysis of these different protein preparations was performed as described by Rakotoarivonina et al. (2002)
except that the antibodies used were the anti-PilA2-C antiserum (dilution 1/2000).
Inhibition of bacterial adhesion.
The effect of the anti-peptide antiserum on the adhesion of R. albus 20 to cellulose was determined as previously described (Rakotoarivonina et al., 2002).
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RESULTS |
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The two ORFs directly downstream of pilA2 in R. albus 8 have significant amino acid identity to SecD and SecF. The SecD-like protein has 34 % (84 of 245 residues) and 29 % (127 of 437 residues) amino acid identity to the Synechocystis sp. protein-export membrane protein SecD and to the SecD component of the Bacillus subtilis SecDF polypeptide, respectively. The SecF-like protein of R. albus has 31 % (91 of 291 residues) amino acid identity to SecF of the B. subtilis SecDF polypeptide. In B. subtilis, the SecD and SecF proteins are fused and, as an integral membrane protein, contribute to the efficiency of protein secretion via the SecYEG translocation channel (Bolhuis et al., 1998). The 468 residue SecD-like and 324 residue SecF-like proteins of R. albus have predicted molecular masses of 50 516 Da and 35 382 Da, respectively. The presence of a
70 promoter sequence upstream of the secD-like gene, but the lack of a prominent promoter sequence upstream of the secF-like gene, suggests that the two genes are cotranscribed. Considering that the translational start codon of the SecF-like protein overlaps the translational stop codon of the SecD-like protein, the SecD- and SecF-like proteins appear to be translationally coupled.
Gene organization downstream of the gp25 gene in R. albus 20 was the same as that in R. albus 8 (Fig. 1). The 1·5 kb of sequenced DNA was found to contain two ORFs in the same orientation as the gp25 gene. The first ORF (468 bp) was preceded by a
70-like promoter sequence and ribosome-binding site and ended with a putative Rho-independent terminator. It encoded a 155 amino acid protein with a calculated molecular mass of 16 844 kDa that shared 98 % identity with PilA2 of R. albus 8 and 41·1 and 41·8 % identity with GP25 (R. albus 20) and CbpC (R. albus 8), respectively. The N terminus of the protein also possessed all the characteristics of Pil proteins, as described above. On the basis of these identities, the gp25 and cbpC genes were renamed pilA1 and the novel downstream gene in both strains was termed pilA2. Although the second ORF (396 bp) is incomplete, it shared 100 % identity with the SecD-like protein identified in R. albus 8, supporting the contention that both strains possess secD/F-like genes in this region of their genomes.
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The pilA1 and pilA2 genes are expressed as both mono- and bicistronic transcripts
We have previously shown that the pilA1 (cbpC) transcript is predominantly monocistronic in R. albus 8 cultured with cellobiose (Pegden et al., 1998). Northern blot analysis of pilA2 expression in R. albus 8 following growth with cellobiose is shown in Fig. 2
. The predominant pilA2-encoding transcript was also monocistronic, but a second, larger transcript was also detected. The estimated size of this second transcript (1·35 kb) is large enough to encode both the PilA1 and PilA2 coding sequences and the RT-PCR results for strain 20 (Fig. 3
) verified the existence of a bicistronic transcript encoding both PilA1 and PilA2 in R. albus 20. When normalized relative to the amount of 16S rRNA present in each sample by phosphorimaging analysis, there were no differences in the abundance of either the mono- or bicistronic transcripts encoding pilA2 following growth of R. albus 8 in EM-cellobiose medium prepared to contain either rumen fluid, PAA/PPA or neither addition (Fig. 2
). We therefore conclude that the pilA2 gene is not conditionally expressed in response to PAA/PPA.
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Transcript abundance for the pilA1 and pilA2 genes was therefore measured by quantitative RT-PCR and calibrated with that of the rrs gene in R. albus 20 and in the mutant D5, following growth to stationary phase on cellobiose (Fig. 4). There were only limited differences between strains 20 and D5 in terms of pilA1 and pilA2 transcript abundance when the strains were cultured with cellobiose, but levels tended to be slightly higher for the mutant strain. Similar to the results obtained with R. albus 8, the amount of pilA1 transcript was 73- and 77-fold higher than that of pilA2 in R. albus 20 and the mutant D5, respectively. Collectively, the results show that the mutant produces similar amounts of pilA1/A2 transcripts as the parent and that the pilA1 transcript is much more abundant than pilA2, although there is evidence for the production of a transcript encoding both the PilA1 and PilA2 ORFs.
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DISCUSSION |
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We previously showed that R. albus elaborates type IV pili on its surface and that these pili mediate bacterial adhesion to cellulose (Pegden et al., 1998; Rakotoarivonina et al., 2002
). Such findings are indeed novel for Gram-positive bacteria, and the genetics and molecular biology underpinning the biogenesis and assembly of these structures is virtually undefined. In R. albus 20, the major pilus subunit is the 25 kDa glycoprotein now referred to as PilA1, and, in R. albus 8, the CbpC protein (Pegden et al., 1998
), which is 73 % identical to PilA1, presumably plays the same role. Accordingly, we propose that CbpC now be referred to as PilA1. Both R. albus 8 and 20 also possess a second gene immediately downstream of pilA1 that encodes a Pil protein (pilA2), followed by genes homologous to secD and secF. This tandem arrangement of pil genes has been described before in several Gram-negative bacteria and in a strain of Synechocystis sp. In Pseudomonas stutzeri, the pilAI gene encodes the major fimbrial subunit, and inactivation of the pilAII gene does not affect fimbrial production but, instead, increases the transformability of P. stutzeri cells, suggesting that this second gene is involved in repression of DNA uptake (Graupner & Wackernagel, 2001
). In Eikenella corrodens, the major fimbrial subunit is encoded by the pilA1 gene, and inactivation of the pilA2 gene does not change the phenotype, indicating that the pilA2 gene in this bacterium is also not essential for the biosynthesis of functionally normal fimbriae. However, when the pilA1 gene was deleted and the pilA2 gene was placed under the control of the pilA1 promoter, E. corrodens produced fimbriae composed of the PilA2 protein (Villar et al., 1999
, 2001
). The cyanobacterium Synechocystis sp. is surrounded by thin and thick pili structures and, while pilA2 gene inactivation does not have any effect on the presence of either type of fimbriae, the thick fimbriae disappear when the pilA1 gene is inactivated (Bhaya et al., 2000
). Using antibodies specific to PilA2, we were able to show that PilA2 is not part of the externalized fimbrial structure but instead is an internalized, membrane-associated protein, as revealed by the presence of PilA2 in both membrane and cytoplasmic fractions of R. albus. In addition, the fact that the anti-PilA2 serum does not inhibit the adhesion of the bacterium to cellulose suggests that PilA2 is not externalized or directly involved in adhesion coordinated by PilA1, similar to the findings outlined above. Considering the cellular localization of the R. albus PilA2 protein and the relative abundance of the pilA2 transcript (and protein) compared with our previous findings for PilA1 (Rakotoarivonina et al., 2002
), the PilA2 protein may be a part of the assembly complex, without being an integral part of the fimbrial structure itself. Several minor, membrane-associated Pil proteins are proposed to form the base structure of the fimbriae in P. aeruginosa (Mattick, 2002
), in a manner similar to the inner-membrane Pil proteins that are a part of the type II (general) protein-secretion pathway in many Gram-negative bacteria (Hobbs & Mattick, 1993
; Pugsley, 1993
). Inactivation of the pilA2 gene in R. albus would permit a determination of its role in fimbrial assembly and function, but, until these bacteria are rendered amenable to experimental techniques in bacterial genetics, this is technically not possible.
We showed the presence of a pilA1pilA2 cotranscript, as well as a monocistronic pilA2 transcript. Our previous study confirmed that pilA1 is also expressed as a monocistronic transcript (Pegden et al., 1998). In this study, pilA1 mRNAs were approximately 70-fold more abundant than pilA2 mRNAs. To explain these results, we can offer two alternative hypotheses: (i) pilA1 and pilA2 genes are cotranscribed and the attenuation of pilA2 expression relative to pilA1 is due to the presence of a predicted hairpin structure in the pilA1pilA2 intergenic region or (ii) pilA2 constitutes a single gene transcriptional unit, with the pilA1 promoter being much stronger than the pilA2 promoter, and transcription starting from pilA1 promoter sometimes goes through the pilA1pilA2 intergenic region, giving rise to a polycistronic message.
The pilA1 and pilA2 gene sequences are identical in R. albus strain 20 and the D5 mutant, indicating that the mutant phenotype cannot be explained by an alteration in these genes. In addition, the fact that the amount of PilA2 protein is similar in the mutant and the parent following growth on cellobiose is in agreement with the pilA2 transcript abundances measured in both strains. On the contrary, although the transcript levels of the pilA1 gene in the parent and mutant strains are similar, PilA1 protein abundance is reduced in the mutant strain D5 (Mosoni & Gaillard-Martinie, 2001). Because of the complex phenotype of the mutant (lack of pili, reduced cellulase activity and altered glycocalyx), we previously assumed that the mutation in strain D5 is pleiotropic in its effects, perhaps within a gene encoding a regulatory function associated with the synthesis or the secretion of surface and/or extracellular molecules. The fact that PilA1 protein is less abundant in the mutant than in the parent but there are only limited differences between the two strains in terms of pilA1 transcript abundance suggests that the mutation exerts its effects at the level of post-translation or secretion.
In conclusion, our initial hypothesis that the pilA1 gene is part of a larger collection of genes involved with the secretion and assembly of type IV pili was supported by the identification of a second Pil-like protein, PilA2, followed by genes encoding accessory functions for protein secretion. Now that the genome sequence for R. albus strain 8 is almost complete, it should be possible to assemble a more holistic picture of the candidate genes involved in the synthesis and assembly of type IV fimbrial proteins in R. albus and to develop a comparative understanding of this mechanism of adhesion for Gram-positive bacteria.
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Received 29 October 2004;
revised 11 January 2005;
accepted 12 January 2005.
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