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1 Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium
2 Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
3 Eurogentec Proteomics GmbH, Warthestraße 21, D-14513 Teltow/Berlin, Germany
4 VAR Ukkel, Groeselenberg 99, B-1180 Brussel, Belgium
Correspondence
Willy Verstraete
willy.verstraete{at}ugent.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The results of gel electrophoresis of aggA PCR amplification products are shown in Supplementary Fig. S1, and an overview of the 11 identified protein spots and their relative spot intensities, and a complete list of all the genes that showed significant upregulation in the COAG strain in at least one sampling event, are shown in Supplementary Tables S1 and S2, respectively, with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and in many cases this process is facilitated by auto-aggregation factors (Schembri et al., 2003a). These factors also allow bacteria to auto-aggregate, a phenomenon readily observed under the microscope as a clumping of cells and, macroscopically, as flocculation and settling of cells from liquid suspensions (Schembri et al., 2001). Different auto-aggregation factors exist in different strains: surface adhesins (Schembri et al., 2003b), type IV pili (Schreiber et al., 2002; Bechet & Blondeau, 2003; Bieber et al., 1998), curli (Olsen et al., 1989) and aggregating adherence fimbriae (Nataro et al., 1993; Czeczulin et al., 1997).
A number of theories have been proposed for the auto-aggregation of micro-organisms, including factors such as substrate gradients, slow growth of the organism, physical/chemical stress, and predation (Farrell & Quilty, 2002). In some cases, auto-aggregation of bacteria can be induced and even become a permanent feature. Pseudomonas putida CP1 exhibits auto-aggregation during the degradation of monochlorophenols and phenol, and it has been shown that this is a response to the toxicity of the added substrates (Farrell & Quilty, 2002). Bossier et al. (2000) observed that after inoculation of the non-aggregative Ralstonia eutropha-like strain AE815 into activated sludge, reisolation on selective medium yielded mutant strain A3, which was characterized by auto-aggregative behaviour.
In our laboratory, an auto-aggregating mutant of Shewanella oneidensis strain MR-1 has been obtained, S. oneidensis COAG, which exhibits increased biofilm formation. S. oneidensis MR-1 is a facultative aerobic Gram-negative bacterium with remarkably diverse respiratory capabilities, including the reduction of several metal ions. As a result, the biological activities of S. oneidensis have considerable implications with regard to direct bioremediation of both metal and organic pollutants (Heidelberg et al., 2002). However, the activities of such a versatile bacterium also have negative effects. In the case of S. oneidensis, unwanted and often hard-to-control effects include the deterioration and destruction of human commodities. For instance, biofilm growth coupled to iron respiration can affect the chemistry of the environment around steel, leading to microbially induced corrosion (MIC). Since S. oneidensis and related Shewanella species have been isolated from corroding steel pipelines (Semple & Westlake, 1987; Dubiel et al., 2002), a mechanistic understanding of the role of Shewanella biofilm formation and metabolic activity may help to facilitate corrosion control (Lee & Newman, 2003). The capacity of S. oneidensis to attach to and detach from solid surfaces and to form biofilms has already been described in a number of applications and studies (Bagge et al., 2001; Little et al., 1998; Thormann et al., 2004, 2005). It has recently been suggested that two type IV pilin gene clusters, MSHA (mannose-sensitive haemagglutinin) and tapABCD, could be involved in this process (Heidelberg et al., 2002), in the same way that MSHA plays a role in biofilm formation by Vibrio cholerae (Chiavelli et al., 2001). This hypothesis is further developed in the work of Thormann et al. (2004), in which it is shown that defects in MSHA type IV pilus biosynthesis and pilus retraction result in severe defects in adhesion to surfaces. Furthermore, motility genes are found to be involved in the three-dimensional development of biofilms. In other studies, exopolymeric substances and surface proteins have been suggested to be involved in transient contact between several Shewanella species and Fe(III)oxides during Fe(III) respiratory activity (Caccavo et al., 1997; Lower et al., 2001).
The goal of this study was to examine the properties of S. oneidensis COAG with regard to biofilm formation and hyper-aggregating behaviour. With proteomics and microarray analysis, surface proteins were screened for their possible relation to the auto-aggregating properties of this strain. By means of insertional mutation, the role of AggA in the auto-aggregation of COAG was elucidated. It was shown that AggA is a necessary auto-aggregation factor for the increased cell-to-cell and cell-to-surface attachment of the hyper-aggregating COAG mutant.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Construction of the insertional mutant AGGA1.
S. oneidensis COAG was used as a parental strain to generate an aggA null allele by integrative disruption with the suicide plasmid pKNOCK-Kmr (Alexeyev, 1999). Briefly, a 768 bp aggA internal fragment was amplified by PCR with primers AGG183F (5'-TCAGGCAATTGCTGGTTACA-3') and AGG951R (5'-ACCACCTACGTTTTGGCTTG-3'). The PCR fragment was digested with SalI and PstI, yielding a 538 bp fragment, which was ligated into the pKNOCK-Kmr vector according to standard procedures (Sambrook & Russell, 2001). The resulting construct was then transformed into Escherichia coli S17-1/
pir and subsequently transferred into S. oneidensis COAG by conjugation. Six transconjugants were picked from LB+Rif (100 mg l–1)+kanamycin (25 mg l–1) agar plates, and disruption of the aggA locus was verified by PCR amplification with primers AGG183F and AGG1122R (5'-GTAGGCATTCCATGCCAGAT-3').
Complementation of the aggA insertional mutation.
S. oneidensis AGGA1, a COAG mutant with an aggA : : pKNOCK-Kmr insertional mutation, was complemented with the pCM62 cloning vector (Marx & Lidstrom, 2001) expressing the aggA gene. Briefly, the complete aggA fragment was amplified from S. oneidensis COAG with primers AGGf (5'-TTTGGATCCTTTTAAGGGCTAAGGACACAGC-3') and AGGr (5'-GCGAATTCGTATGGAGCAACCCGCTAAA-3'). Both primers were provided with the recognition sequence for BamHI and EcoRI. The aggA PCR fragment was ligated (sticky end) into the pCM62 vector according to standard procedures (Sambrook & Russell, 2001). The resulting vector, pCM62, with a 1510 bp BamHI–EcoRI DNA fragment containing the aggA gene from S. oneidensis COAG, was designated pAG11. This construct was then transformed into E. coli S17-1/pir and subsequently transferred into S. oneidensis AGGA1 by conjugation. Six transconjugants were picked from LB+Rif (100 mg l–1)+kanamycin (25 mg l–1)+tetracycline (12·5 mg l–1)+X-Gal (40 µg ml–1)+IPTG (1 mM) agar plates, and complementation with aggA was verified by PCR amplification with primers AGGf and AGGr. The results of gel electrophoresis of the PCR products are shown in Supplementary Fig. S1. Expression of aggA in the complemented AGGA1(pAG11) was verified by RT-PCR of extracted mRNA with internal aggA primers AGGA617F (5'-GGGCTAATGCGAATGTGATT-3') and AGGA951R (5'-ACCACCTACGTTTTGGCTTG-3') according to the manufacturer's instructions (Qiagen) and standard procedures (Sambrook & Russell, 2001).
Determination of aggregation index.
Determination of the aggregation index (AI) was done according to Malik & Kakii (2003). Briefly, 10 ml of an S. oneidensis culture in M9 medium was rigorously vortexed to destroy aggregates, and the ODtotal of this suspension was determined. Another 10 ml of the culture was centrifuged, without vortex treatment, for 2 min at 650 g, providing ODsupernatant.
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Enzymic treatment of the aggregates.
S. oneidensis COAG was grown in culture flasks containing 50 ml LB+100 mg Rif l–1. Enzymic treatments were repeated in triplicate. All enzymes except alcalase were added in a 1 : 250 (w/w) enzyme : cell dry weight ratio, and incubation with the aggregates was at 37 °C for 3 h with shaking (150 r.p.m.) at neutral pH. Alcalase was added at a concentration of 0·06 AU l–1, according to the manufacturer's instructions (ORFFA Health and Nutrition, Londerzeel, Belgium). As a control, a treatment with an inactive proteolytic enzyme, pepsin at neutral pH, was carried out under identical incubation conditions.
Flow-cell experiments.
Biofilms were established in 1/10 LB medium, maintaining the original NaCl concentration of 5 g l–1, at 25 °C on a glass carrier (glass flow-through tubes, internal diameter 3 mm, external diameter 4 mm), using a twelve-channel autoclaved flow-cell system. In the case of the complemented AGGA1(pAG11) mutant, X-Gal (40 mg l–1) and IPTG (1 mM) were added to the medium. The tubing consisted of marprene tubing (Watson-Marlow, Brussels, Belgium) in the pump head and Masterflex 96400-16 silicone tubing (Cole-Parmer Instrument Co. Ltd, London, UK) in the flow-cell system. The flow cell was inoculated with 1 ml of an overnight LB culture at OD610 1·5. One hour of stagnation was applied to allow initial adhesion of the bacterial cells onto the glass. After initial attachment of the cells, the medium was pumped through the flow channels at a rate of 0·7 ml min–1 using a peristaltic pump (Watson-Marlow). The biofilm was sampled in the early development stage (6 h) and as a mature biofilm (50 h). The glass biofilm carrier was homogenized and the attached biofilm was suspended in 0·85 % saline. A dilution series was plated on LB agar plates with appropriate antibiotic selections, and c.f.u was calculated per square millimetre of carrier material. Every biofilm sample was obtained from an independent experiment in triplicate.
Microscopy of the aggregates.
Aggregates were analysed by standard phase-contrast microscopy on a Zeiss Axioskop II microscope. The microscope was equipped with a Peltier cooled single-chip digital colour charge-coupled device (CCD) camera (Hamamatsu) connected to a PC to obtain digital images.
Microarray experimental design.
S. oneidensis COAG cultures were set up in triplicate (n=3) in 1000 ml culture flasks with 150 ml LB broth, and aerobic conditions were maintained by shaking at 100 r.p.m. The transcriptional profile of the COAG cells in these cultures was compared to that of S. oneidensis MR-1, grown under identical conditions, at several time-points. There were four sampling events, at intervals of at least 2 h. The first samples were taken in the early exponential phase (OD 0·5) from both S. oneidensis MR-1 and COAG cultures, and this sampling event was termed t0. Consecutive samples were taken at 2, 4 and 8 h after t0, and referred to as sampling events t2, t4 and t8, respectively. Gene expression analysis was performed using three independent microarray experiments with fluorescent dye reversal, each slide containing two replicate arrays, and at four sample-taking events, yielding a maximum of 48 data points per gene.
RNA isolation and preparation of fluorescein-labelled cDNA.
Cells of both wild-type and COAG strains of S. oneidensis were harvested by centrifugation, and total cellular RNA was isolated using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA samples were treated with RNase-free DNase I (Ambion) to digest residual chromosomal DNA, and then purified with the Qiagen RNeasy Mini kit prior to spectrophotometric quantification at 260 and 280 nm. Fluorescein-labelled cDNA copies of total cellular RNA extracted from wild-type and mutant cells were prepared essentially as described previously (Thompson et al., 2002). Two sets of duplicate reactions were carried out in which the fluorescent dyes were reversed during cDNA synthesis to minimize gene-specific dye effects. The labelled cDNA probe was purified and concentrated as described previously (Thompson et al., 2002).
Microarray hybridization and scanning.
The PCR product-based whole-genome ORF arrays for S. oneidensis MR-1 were constructed as described previously (Gao et al., 2004).
Quantitative analysis of hybridization intensities and normalization.
TIFF scanned images (16-bit) were analysed with the software package ImaGene 5.5 (Biodiscovery Inc., Los Angeles, CA) to determine spot intensities. Noise and spots of poor signal quality were removed from the data matrix (Hegde et al., 2000). Data in this output were normalized (LOWESS) with the software package Genespring 6.0 (Silicon Genetics, Redwood City, CA). Data were subjected to two filters: a filter on confidence, selecting only significant differences between data points of wild-type and mutant as determined by a Student's t test (P
0·05), and a filter selecting only gene expression values with a greater than twofold ratio between the two fluorescent signals (Schena et al., 1996).
Two-dimensional (2D) PAGE of membrane-enriched preparations of S. oneidensis.
In order to generate membrane-enriched preparations of S. oneidensis, pellets of MR-1 and COAG, harvested from an aerobic LB culture in the mid-exponential growth phase, were sonicated for 3x30 s in carbonate buffer on ice (Molloy et al., 1998). The samples were diluted and stirred on ice for an additional 3 h. Membranes were collected by ultracentrifugation at 4 °C. Pellets were resuspended in water and recentrifuged at 4 °C. This washing step was repeated two additional times. The final pellet was resuspended in detergent-containing solubilization buffer (0·1 % Triton X-100) and treated by sonication on ice to enhance solubilization. Insoluble particles were then removed by centrifugation, and the cleared supernatant was treated with tributyl phosphine (TBP) and iodoacetamide before loading of the samples onto high-resolution non-equilibrium pH gradient electrophoresis (NEPHGE) first-dimension gels. Total protein (120 µg per gel, as determined by a protein assay using BSA as reference) was used for triplicate gels of each sample. For the second dimension, glass-plate-immobilized 15 % SDS-PAGE gels (20x30 cm) were used. After electrophoresis, the gels were stained with SYPRO Ruby (Invitrogen).
Image acquisition and analysis of 2D gels.
SYPRO Ruby-stained gels were scanned using a Fuji FLA-3000 fluorescence scanner (Raytest, Straubenhardt, Germany). Pictures were imported into the 2D evaluation software Melanie 3 (Genebio, Geneva, Switzerland), matched spots were analysed for significant intensity differences, and COAG upregulated (Student's t test value 
0·95) spots were selected.
Protein identification by mass spectrometry.
Selected protein spots were picked using an Ettan spot picker robot (Amersham Biosciences) and digested with trypsin. For desalting and matrix crystallization, a GYROS SP1 micro CD system (GYROS AB, Uppsala, Sweden) was used. Mass fingerprints were used to search the NCBI database using MASCOT (Matrix Science Ltd, London, UK) and identify the proteins.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The AI represents the fraction of cells associated in aggregates. Aggregates started to form in the early exponential phase, after which the aggregate size steadily increased, and aggregates remained present in the late stationary phase. This was confirmed by microscopic and visual examination. The AI of the S. oneidensis MR-1 wild-type was determined and was found to be below 0·01 for more than 24 h. The following proteolytic enzymes were added to a stationary-phase COAG culture in LB broth and lowered the AI significantly (Fig. 1b): proteinase K, of the serine proteases 
-chymotrypsin and trypsin, neutrase, alcalase, and a commercial protease mix. A phase-contrast image of COAG aggregates is shown in Fig. 2.
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) was used to monitor the altered gene expression of COAG with respect to MR-1. For each condition, a total of six independent hybridizations were performed, including three biological replicate experiments with fluorescent-dye reversal. Following spot intensity quantification and data normalization, 249 genes showed significant upregulation in COAG grown aerobically in LB broth, in at least one sampling event (mean fold change 2·0 and P0·05). An overview of the 11 identified protein spots and their relative spot intensities, and a complete list of all the genes that showed significant upregulation in COAG in at least one sampling event, are shown in Supplementary Tables S1 and S2, respectively.
Only upregulated outer-membrane proteins for which the increased expression of the corresponding genes was confirmed by microarray analysis were considered in this study. The gene encoding the agglutination protein AggA, part of the putative RTX toxin operon, was overexpressed in COAG, and this was confirmed by 2D PAGE, in which induction of the three monomers of this protein in COAG was observed (Tables 1 and 2). The transcription study revealed that the other genes in the RTX operon were significantly upregulated in COAG as well. The SO4317 gene, encoding a putative RTX toxin protein, was significantly overexpressed at all sampling time-points. The proposed components of the secretion system for this putative RTX protein, consisting of rtxB (SO4318), encoding an ATP-binding protein in the inner membrane, SO4319, encoding an HlyD-family membrane-fusion protein, and aggA (SO4320), encoding the outer-membrane component of the secretory system, were all found to be upregulated in the auto-aggregating mutant (Table 1). The expression levels of the outer-membrane component aggA steadily increased at consecutive sampling time-points. The upregulation of rtxB and SO4319 was found to be significant (mean fold-change 2 and P0·05) at the earlier growth stages (t0 and t2).
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), and the transcription of mtrB was found to be increased up to 4·3-fold (Table 1).
Analysis of the aggA mutant strain
To establish the role of AggA in hyper-aggregation and increased biofilm formation, the aggA gene was inactivated in COAG by integrative disruption with the suicide plasmid pKNOCK-Kmr. Disruption of the aggA locus in six transconjugants was verified by PCR amplification with primers AGG183F and AGG1122R. A product of approximately 940 bp was amplified from the parental COAG DNA, whereas from all the transconjugant aggA : : pKNOCK-Kmr DNA, a 3·3 kb product was amplified, confirming the integration of the plasmid into the aggA gene. Supplementary Figure S1 illustrates the agarose gel loaded with the different PCR products. The aggregating property of the S. oneidensis COAG transconjugants with aggA integrative disruption was analysed by microscopy and the AI was determined for all of them. For all transconjugants, the hyper-aggregating property was completely lost, and this was due to insertional mutation of aggA in COAG. The AI of the aggA insertion mutant was not significantly different from that of the MR-1 wild-type. From these results, it follows that the AggA agglutination protein plays a central role in the aggregation by COAG.
Biofilm formation
COAG was able to form a dense biofilm on a glass carrier within 6 h, and its biofilm-forming capacity was considerably higher than that of MR-1. Even a mature MR-1 biofilm did not reach this density (Fig. 3). After 50 h, COAG biofilms consisted of (1·5±0·1)x106 c.f.u. mm–2, whereas MR-1 biofilms only reached a cell density of (4·4±0·6)x104 c.f.u. mm–2. Biofilm formation was analysed for one of the S. oneidensis COAG transconjugants with aggA integrative disruption, designated AGGA1, in a flow-cell set-up with three independent replicates. Due to an insertional mutation in the aggA locus, AGGA1 had lost the increased biofilm-forming properties of COAG. In the early biofilm development phase (6 h), in which initial cell-to-surface attachment takes place, AGGA1 biofilm density was significantly lower than that of both MR-1 and COAG. After 50 h, the cell density in AGGA1 biofilms had reached that of MR-1 biofilms. By complementation of the insertional mutation in AGGA1 with the cloning vector pCM62 expressing the aggA gene from COAG, the strong inhibition of initial cell-to-surface attachment could be nullified. The complemented AGGA1(pAG11) mutant exhibited a biofilm cell density of (1·3±0·03)x104 c.f.u. mm–2 within 6 h, and this corresponded well to the biofilm-forming capacity of MR-1.
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DISCUSSION |
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). As a consequence, the hyper-aggregating phenotype could be accompanied by changes in a large collection of genes, which may or may not be related to the auto-aggregating property. Therefore, the microarray and proteomics analyses have been focused on altered expression of outer-membrane proteins and proteins directly associated with them, in order to identify candidate surface proteins that could be related to the hyper-aggregation and increased biofilm formation of S. oneidensis COAG.
The role of surface-located proteins in the process of COAG auto-aggregation, and in the formation of organized multicellular structures in general, was confirmed by different proteolytic treatments of the aggregates. All of the proteolytic enzymes tested resulted in deflocculation within 3 h of incubation. In a similar test, Malik et al. (2003) have studied the effect of the addition of trypsin and actinase E on aggregate structure, and have found that protein structures mediate co-aggregation between Acinetobacter johnsonii and Microbacterium esteraromaticum. To further substantiate the involvement of surface-located proteins in COAG aggregation, we investigated differential expression of cell membrane proteins by 2D PAGE of membrane-enriched preparations of COAG and MR-1. The results from the proteomics analysis were compared to those of COAG and MR-1 differential gene expression observed by transcriptional analysis with DNA microarrays. The predicted transport component of a putative RTX toxin operon, agglutination protein AggA, was found to be upregulated in COAG. DNA microarray analysis confirmed that all genes from this operon were upregulated in COAG. The role of other aggregation factors, such as type IV-pili, which have been suggested by Heidelberg et al. (2002) to be important during S. oneidensis attachment to solid surfaces, could not be established in our study, since no significant difference was observed with regard to these structures in the parent and mutant (results not shown). In Pseudomonas fluorescens, a similar ATP-binding cassette (ABC) transporter system for the secretion of a large protein has been reported to be involved in biofilm formation (Hinsa et al., 2003). For the latter organism, the putative lapEBC-encoded ABC transporter participates in the secretion of the large protein LapA (large adhesion protein A), and this system is required for transition from reversible to irreversible attachment during biofilm formation. LapB is the predicted inner-membrane protein of 74 kDa, analogous to the predicted 79 kDa RtxB ATP-binding protein of S. oneidensis. RtxB is proposed to be the inner-membrane-anchored ATPase of an ABC transporter involved in export of the putative RTX toxin. In P. fluorescens, LapC has been described as an HlyD-family membrane-fusion protein, part of the proposed ABC transporter, and in S. oneidensis, the SO4319 gene product is predicted to be an HlyD-family secretion protein as well. Finally, the predicted P. fluorescens outer-membrane protein LapE, with sequence similarity to the P. putida AggA adhesin, has been suggested to promote stable adhesion of P. fluorescens, possibly in association with LapA. This is comparable to our observation that overexpression of the S. oneidensis AggA outer-membrane protein was associated with the altered cell-adhesion properties of COAG.
To further establish the importance of AggA in COAG aggregation and increased biofilm formation, the effect of insertional inactivation of the aggA gene on the phenotypic properties under aerobic growth conditions was investigated. The aggA mutation reversed the AGGA1 phenotype to that of the original MR-1 strain, with loss of hyper-aggregation and the reacquisition of a biofilm-forming capability that was not significantly higher than that of the MR-1 wild-type. Interestingly, the initial cell density was significantly lower in AGGA1 biofilms than in both MR-1 and COAG biofilms. This indicates the importance of AggA in the initial phase of biofilm formation, cell-to-surface attachment. A short period of exposure of COAG to proteolytic enzymes resulted in digestion of the outer-membrane agglutination protein, and this also resulted in loss of aggregation. These experiments indicate the strong involvement of the upregulated aggA gene and the associated putative RTX operon in COAG hyper-aggregation, and the necessity of the AggA outer-membrane protein to the COAG phenotype with its increased adhesive properties. These findings were further confirmed by complementation of the AGGA1 mutant with pCM62 expressing the aggA gene from COAG. In this cloning vector, aggA was under the control of a lacZ promoter, and as such could not be upregulated in the same manner as in COAG, but the inhibitory effect of the aggA null allele in mutant strain AGGA1 on the initial phase of biofilm formation could nevertheless be eliminated by this complementation. Biofilm-forming capacity was restored to the level of the MR-1 wild-type. Thus, it was shown that auto-aggregation by S. oneidensis COAG, coupled to increased biofilm formation at the stages of both initial surface attachment and biofilm maturation, requires upregulation of the AggA agglutination protein. The AggA outer-membrane protein controls cell-to-surface attachment in S. oneidensis, since insertional mutation of the aggA gene strongly inhibited the initial surface colonization, but biofilm formation could be restored to the level of the MR-1 wild-type by complementation with a cloning vector expressing the aggA gene under the control of a lacZ promoter. The auto-aggregating phenotype could not be restored by this complementation, and required AggA overexpression.
A recent study has suggested the involvement of MSHA, the mannose-sensitive haemagglutinin type IV pilus, during the initial adhesion of S. oneidensis to abiotic surfaces, observed after 1 and 4 h (Thormann et al., 2004). Also, disruptions of motility genes (flhB, fliK and pomA) and of a gene involved in pilus retraction (pilT) affect biofilm formation and the progression of biofilm development into a pronounced three-dimensional architecture. Although none of these elements was found to be upregulated in S. oneidensis COAG, it is possible that the three-dimensional development of COAG biofilms is under the influence of several of these factors. Our data suggest, however, that during initial attachment, other outer-membrane factors are involved as well. Although initial attachment by S. oneidensis COAG was found to be strongly inhibited by aggA disruption, the mature biofilm nevertheless developed with a decreased density. It is possible that other surface structures described in the study of Thormann et al. (2004) may have substituted for part of the role of AggA in this process.
The characterization of the adhesive properties of COAG provided new insights into cell-to-cell and cell-to-surface attachment by S. oneidensis. The importance of AggA in biofilm formation by the S. oneidensis MR-1 wild-type has, to our knowledge, not been described in the literature. However, it was shown in our research that AggA is a necessary factor for the increased surface and cell adhesion of the hyper-aggregating COAG mutant, and that cell-to-surface attachment can be inhibited and restored by, respectively, disruption and complementation of aggA. Similarly, the MSCRAMM (microbial surface components that recognize adhesive matrix molecules) adhesin LapA is a cell-surface protein that enables P. fluorescens to attach to plastic, glass and quartz sand (Hinsa et al., 2003). In P. putida, the lapA gene is of importance during attachment to plastics, glass and corn seeds (Espinosa-Urgel et al., 2000). The similarities between the function and ABC-transporter structure of the operons containing aggA and lapA, coupled to the necessity of AggA to the increased adhesive properties of COAG, the aggA upregulation in COAG, and the control that can be exerted over the initial stages of biofilm formation by disruption and complementation of aggA, strongly suggest that the MR-1 wild-type also partially depends on aggA, and possibly the putative RTX operon, for attachment to surfaces and biofilm formation. Further research is required to formulate any definite statements about MR-1, although recent research has shown that AggA is overexpressed in biofilms of the MR-1 wild-type compared to MR-1 planktonic cells (De Vriendt et al., 2005).
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ACKNOWLEDGEMENTS |
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Received 18 May 2005;
revised 31 October 2005;
accepted 8 November 2005.
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) was also measured at every sampling. All measurements were done independently and in triplicate. The AI of MR-1 wild-type did not exceed 0·01 over 24 h. (b) Effect of digestion by different enzyme preparations on the stability of S. oneidensis COAG aggregates. Conditions appropriate to each enzyme were employed. Inactive pepsin served as a negative control. Black bars, AI before treatment; grey bars, AI after treatment.
