1 Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada V6T 1Z3
2 Department of Microbiology, Montana State University, Bozeman, MT 59717, USA
3 Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902, USA
4 Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, USA
Correspondence
Michael J. Franklin
umbfm{at}montana.edu
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ABSTRACT |
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A figure showing 2DGE of protein extracts is available as supplementary data with the online version of this paper.
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INTRODUCTION |
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In marine environments, calcium is an element that is often enriched on surfaces, either as sedimentary calcium deposits or in association with other marine organisms. Therefore, calcium may be one of the factors that bacteria sense during biofilm-associated growth. Calcium is known to be important for bacterial biofilm formation (see Geesey et al., 2000 for a review). In particular, calcium is involved in specific and non-specific interactions between cells and the substrata (Craven & Williams, 1998
; Kallstrom et al., 2000
; Matsumoto et al., 2000
; Waligora et al., 1999
). Calcium-binding proteins are often involved in bacterial adhesion to a surface, and can be important for cellcell aggregation (Espinosa-Urgel et al., 2000
; Hinsa et al., 2003
; Rose, 2000
). Calcium is also important as an ionic cross-bridging molecule for negatively charged bacterial polysaccharides. Kierek & Watnick (2003)
demonstrated that Ca2+ plays a role in the architecture of Vibrio cholerae biofilms. In particular, when Ca2+ was removed from the medium, as may occur when cells are first exposed to a marine environment then switched to a freshwater system, the biofilms disintegrate (Kierek & Watnick, 2003
). The results demonstrated that Ca2+ is important for the ionic cross-bridging of the O-antigen polysaccharide in the biofilm matrix of V. cholerae.
This role of Ca2+ has been established (Geesey et al., 2000; Kierek & Watnick, 2003
); however, Ca2+ may also play a signalling role in bacterial gene expression, particularly during biofilm-associated growth where calcium concentrations may be elevated. Calcium is an important regulatory molecule in eukaryotic cells, but less is known about its role as a regulator in prokaryotes. There is evidence for calcium-mediated regulation in bacteria, including calcium regulation of channels and transporters (Holland et al., 1999
; Michiels et al., 2002
; Norris et al., 1996
). In addition, calmodulin-like proteins are widespread in prokaryotes (Yang, 2001
). In Pseudomonas aeruginosa, Ca2+ acts as a regulatory molecule, and influences the production of extracellular enzymes and toxins, including extracellular proteases that increase at high [Ca2+], and toxins secreted by the Type III secretion system that are repressed by high [Ca2+]. In addition, the amount of extracellular polysaccharide material of an alginate-producing strain of Pseudomonas aeruginosa is induced as much as eightfold by Ca2+ (Sarkisova et al., 2005
). Therefore, Ca2+ may have dual roles in biofilm formation, as both an ionic cross-bridging molecule for matrix materials and a sensory ion for gene expression of biofilm-associated components.
In this study, we tested the effect of Ca2+ on a marine Pseudoalteromonas sp. isolate during biofilm-associated growth. Pseudoalteromonas spp. are bacteria found in high numbers in a variety of marine environments (Skovhus et al., 2004). In addition to free-swimming cells, Pseudoalteromonas spp. are often found attached to surfaces, including inanimate objects and other marine organisms (Egan et al., 2000
; Holmstrom & Kjelleberg, 1999
). Since Pseudoalteromonads successfully inhabit many marine environments, including biofilms, it is likely that they respond to multiple signals during changes in environmental conditions, such as those that would occur during biofilm formation and development.
Several experimental approaches have been used to characterize the regulatory changes that occur in bacteria during biofilm development. These approaches have included: microarray analyses (Ren et al., 2004; Schoolnik et al., 2001
; Whiteley et al., 2001
; Zhu & Mekalanos, 2003
), in vivo expression technology (Finelli et al., 2003
), mRNA subtractive hybridization (Sauer & Camper, 2001
), transposon mutagenesis studies (O'Toole & Kolter, 1998
) and proteomic studies (Sauer & Camper, 2001
; Sauer et al., 2002
). Advances in two-dimensional gel electrophoresis (2DGE) allow high resolution of protein spots, and cluster analysis of the protein profiles provides information on global changes of protein amounts following changes in environmental conditions. Proteomic technologies are particularly useful for analysis of uncharacterized bacteria, since these technologies do not require a genetic system for manipulating the bacterial DNA. In addition, although useful, a complete genome sequence is not necessary for proteomic studies. Therefore, proteomic approaches can be useful for studying recent environmental isolates, such as the marine bacterium characterized here. The goal of this study was to characterize the physiological changes that occur in a marine isolate, Pseudoalteromonas sp. 1398, during biofilm formation, and to determine if the bacteria respond to calcium during a switch from planktonic growth to biofilm formation.
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METHODS |
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Minimal marine medium (MMM) used in these studies was composed of the following (per litre): 18 g NaCl, 5 g MgSO4, 1 g NH4Cl, 0·6 g KCl, 1 g glucose, 5 mg K2HPO4, 0·05 g NaNO3, 0·15 g NaSiO3.9H2O, 8 ml Tris buffer (1·45 M Tris/HCl, 0·55 M Tris Base, pH 7·6), 10 µl trace metals solution, 1 ml vitamins solution. Trace metals solution contained (per litre): 380 mg FeCl3.6H2O, 430 mg MnCl2.4H2O, 2 mg CoCl2.6H2O, 66 mg ZnSO4.7H2O, 340 mg H3BO3, 0·37 mg CuSO4.5H2O, 3 g Na2EDTA. Vitamins solution contained (per litre): 0·5 g thiamine, 1 mg biotin, 0·2 mg vitamin B12. Stock cultures were maintained on rich medium, which contained (per litre): 16·7 g NaCl, 5 g MgSO4, 1 g NH4Cl, 0·6 g KCl, 1 g glucose, 5 g yeast extract, 10 g tryptone. Basal salts medium was composed of the following (per litre): 18 g NaCl, 5 g MgSO4, 0·6 g KCl. CaCl2 was added to the media to produce final concentrations of 0·25, 1·0, 5·0 and 10 mM.
Growth analysis of planktonic and biofilm cultures.
For analysis of planktonic cells, bacteria were grown in MMM in culture flasks (1 : 100 inoculum) with shaking (250 r.p.m.) at room temperature. Measurements of turbidity (OD500) were taken every 2 h, and the amount of total cellular protein was determined every 6 h.
Biofilm growth experiments were performed with biofilms cultivated in flowing systems on hydrophobic or hydrophilic surfaces that included Teflon mesh, glass coverslips and silicon tubing. Biofilm growth on Teflon mesh was performed in once flow-through 1 litre bioreactors. Planktonic cultures (700 ml of an 18 h culture) were used to inoculate the reactor containing the mesh. Cells were allowed to attach for 60 min, and then the cell suspension was replaced with fresh minimal medium. Flow of fresh medium was initiated at 2 ml min1. For biomass analysis, the mesh was removed from the reactor, and washed twice in marine salts buffer (MMM without glucose) to remove planktonic and loosely adhered cells. Attached cells were then removed by ultrasonication with a probe-type cell-disruptor (Cole Parmer Instruments) for 3 min, with intermittent ultrasonication cycles of 10 s on and 10 s off. The amount of total cellular protein for attached bacteria was determined per unit surface area by using the bicinchoninic acid (BCA) assay (Pierce Biotechnology). Biofilm growth on glass coverslips was determined by using the once flow-through system as described previously (Nivens et al., 2001). The flow-cell system consisted of a medium reservoir, pump, silicon tubing, a flow-cell and a waste container. The flow-cell contained a glass coverslip (5·3 cm2) as the substratum, which was sealed to a polycarbonate support with a Viton gasket. Biofilms were removed from the surface and analysed as described for the Teflon mesh experiments. Biofilms were also cultivated on silicon tubing (size 18 Masterflex tubing 3060 cm) as described by Sauer & Camper (2001)
. The silicone tubing was filled with marine salts buffer, and the biofilms extracted with the plunger of a 5 ml syringe. Biofilms in each reactor type were cultivated for 7 to 106 h at room temperature.
Microscopic analysis of biofilms.
Scanning confocal laser microscopy (SCLM) and light microscopy experiments were performed on biofilms cultivated on glass coverslips using the once flow-through system (Nivens et al., 2001). Stacks of confocal images (512x512 bit resolution) were collected using a Leica TCS NT SCLM equipped with a x100 objective. Bacteria were observed following staining of the cells for 20 min with LIVE/DEAD BacLight stain (Molecular Probes). Three-dimensional reconstructions of the images were made using a maximum projection of the image stacks. The total surface-associated biomass was observed with reflected laser light of combined 488 and 586 nm wavelengths. Biofilms were stained with alcian blue (0·1 % in water), and the extracellular acidic polysaccharide surrounding the bacteria (Gerhardt, 1994
) observed with a Olympus PH2-RFCA light microscope with x100 objective. Transmission electron microscopy (TEM) was performed on 18 h planktonic cultures and on biofilms cultivated on silicone tubing (106 h biofilms). For TEM, bacteria were fixed with 25 % glutaraldehyde, applied to 300 mesh Formvar coated cooper grids, stained with 1 % uranyl acetate and analysed with a Zeiss 100CA transmission electron microscope.
Extracellular protein analysis.
Extracellular proteins were characterized from 18 h planktonic cultures or from biofilms (106 h on silicone tubing). Cells were harvested from the walls of the tubing as described above, and removed from the medium by centrifugation (twice for 15 min at 10 000 g). Proteins from the supernatants were precipitated by adding ice-cold trichloroacetic acid (15 % final concentration) followed by incubation on ice for 1 h. TCA-precipitated extracellular proteins were pelleted by centrifugation, and the pellets were washed twice with ice-cold acetone. Protein pellets were resuspended in solubilization buffer (8 M urea, 2 M thiourea, 35 mM DTT, 2 % SDS, 1 mM EDTA, 10 mM Tris/HCl pH 8·0). The protein concentrations were determined by a modified BCA method (Pierce). Protein samples (5 µg) were resolved by 12 % SDS-PAGE (Laemmli, 1970). Proteins were stained with either silver nitrate or Coomassie blue. Immunoblotting was used for flagellin determinations. Proteins were electroblotted onto nitrocellulose membranes and probed with antibodies against the flagellin of Pseudomonas aeruginosa strains PAK and PAO1 (generously provided by Dr Daniel Wozniak, Wake Forest University, Winston-Salem, NC, USA). Horseradish-peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody, and flagellin bands were detected using chemiluminescence (Ausubel et al., 1988
).
2DGE.
Following removal of cells from the Teflon surface as described above, cells were centrifuged and the cell pellets were resuspended in 200400 µl TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8·0), containing 0·3 mg PMSF ml1. The cells were disrupted by ultrasonication for 2 min with 10 s on and 10 s off cycles. Unbroken cells and debris were removed by centrifugation at 15 000 r.p.m. for 30 min, and cell-free crude protein extracts were either stored at 20 °C or used immediately for further studies. Protein concentrations were determined by BCA assay (Pierce).
Protein 2DGE was performed according to published procedures (Gorg et al., 1995, 2000
; Gorg, 1999
; Rabilloud, 2000
) as modified by Sauer et al. (2002)
. The first dimension of protein separation was carried out using Immobiline IPG (immobilized pH gradient) DryStrips (18 cm, pH 47, linear; Amersham). The IPG strips were rehydrated with the protein sample (100700 µg total protein) in 450 µl rehydration solution (11 M urea, 2·7 M thiourea, 35 mM DTT, 3 % CHAPS, pharmalyte pH 310). The strips were incubated overnight at room temperature under mineral oil. After rehydration, IEF was carried out for a total of 35 kVh at 20 °C, under mineral oil, using a Multiphor system (Amersham Biosciences). Following this, the IPG strips were equilibrated in DTT and iodoacetamide equilibration buffers (6 M urea, 0·5 M Tris/HCl, 30 % glycerol, 4 % SDS, pH 6·8), and layered onto 10 % SDS-PAGE gels electrophoresed using a DALT12 system (Amersham Biosciences). Molecular mass markers (Bio-Rad) were run in the same gel, or in a parallel gel under identical conditions. Protein spots were detected by staining with silver nitrate or colloidal Coomassie blue G250.
Cluster analysis of protein spots.
Differential analysis of 2DGE protein maps was performed with the Progenesis (Nonlinear Dynamics) software package. This software detects protein spots, subtracts the background and assigns signal intensity value to each spot. Signal intensities were normalized against the total signal intensity of all the spots on a gel multiplied by 100. The corresponding spots were matched between the gels using the Progenesis combined warping and matching algorithm. Spots with a normalized volume above 0·02 and averaged over two gels (from independent growth experiments) were recorded. Cluster analysis was performed by using data files generated by Progenesis and exported to Microsoft Excel. Plots of the log10 values for each spot signal intensity were performed using GeneSpring software (Silicon Genetics).
Protein identification.
N-terminal sequencing and tandem mass spectrometry (MS/MS) were used for the identification of proteins. For N-terminal sequencing, the gels were equilibrated for 20 min in blot buffer (according to the manufacturer's guidelines; Hoefer Dalton) and the proteins electroblotted onto Immobilon-P PVDF membranes (Millipore). Electroblotting was carried out for 2 h at 300 mA constant current in a Hoefer Dalton Trans-Blot unit. The PVDF membranes were stained with 0·1 % Coomassie blue G250 in 50 % methanol/7 % acetic acid for 5 min, and destained with 50 % methanol. The membranes were washed with water and allowed to dry before storage at 20 °C. The protein spots of interest were excised from the membranes, and N-terminal amino acid sequenced by Midwest Analytical (http://www.mastl.com), using the Edman degradation method. Protein identification was performed using a protein BLAST search for short nearly exact sequences at http://www.ncbi.nlm.nih.gov/blast (Altschul et al., 1997). For MS/MS analysis, the spots of interest were excised from Coomassie blue stained gels and trypsin digested. MS/MS analysis was performed by Proteomics Centre, Genome BC, University of Victoria (www.proteincentre.com), using a Sciex linear ion trap quadrupole LC-MS/MS mass spectrometer (Applied Biosystems). Protein identification of MS/MS data was performed by using BLAST at http://dove.embl-heidelberg.de/Blast2/msblast.html (Shevchenko et al., 2001
).
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RESULTS |
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Similar results for the amount of exopolysaccharide were observed with TEM of the bacteria (Fig. 3). In planktonic culture, the cells cultured at low [Ca2+] had single polar flagella, and did not appear to synthesize detectable levels of extracellular matrix material (Fig. 3A
). At 10 mM CaCl2, the planktonic cells appeared to lose their flagella, and instead produced material that was probably polysaccharide (Fig. 3B
). When grown in biofilm at low [Ca2+], the bacteria produced small amounts of extracellular material (Fig. 3C
). The amount of this matrix material increased at higher concentrations of calcium (Fig. 3D
), confirming the alcian blue staining results. No flagella were associated with the cells cultivated in biofilms (although some broken flagella were observed as debris in these cultures).
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Calcium affects the global proteomic response of biofilm-associated Pseudoalteromonas sp. 1398, and has less effect on planktonic cells
Since Ca2+ affects biofilm formation and extracellular product formation by this bacterium, we also determined if it influences the amounts of cytosolic proteins. For these studies, we first evaluated the proteomic responses of Pseudoalteromonas sp. at a baseline [Ca2+] (1·0 mM) by 2DGE. The bacteria were cultivated in planktonic culture for 18 h, and therefore their proteomes contain proteins produced up to and through the exponential phase of growth. Following planktonic growth, the bacteria were allowed to colonize a Teflon surface for 7 h. A comparison of these proteome maps reflects the changes in the physiology of the bacterium when shifted from planktonic growth to the early stage of biofilm development. A 2DGE differential analysis of cytosolic proteins is shown in Fig. 5(A). Differential-comparison analysis of the protein profiles was obtained using Progenesis and GeneSpring software, and Fig. 5(B)
shows the distribution of the resultant ratios (log10) for each protein resolved on the 2D gels, for the mean of duplicate samples. The separation lines show the border between the spots with different expression profiles, and the spots along the x- and y-axis are those found only in the planktonic or sessile biofilm culture, respectively. On average, 800 protein spots were resolved per gel. Of the 800 spots, approximately 200 showed a twofold or greater differential intensities in biofilm versus planktonic cultures under this growth condition (1 mM Ca2+). These include 93 proteins showing increased abundance in biofilms, 47 showing decreased abundance in biofilms and 62 that appear only in the biofilm culture. These differences represent approximately 25 % of the proteins resolved by 2DGE, and demonstrate that Pseudoalteromonas sp. cells undergo major physiological changes during a shift from planktonic to biofilm culture.
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Twenty-four proteins were grouped as class III, those with decreased intensity in biofilms, but unaffected by Ca2+ (example shown in the Supplementary Figure E1E4 and F1F4, labelled spots 14 and 15). N-terminal peptide sequences of protein 14, gave strong identity to a protein in peroxiredoxin/glutaredoxin family (Heidelberg et al., 2000). The Supplementary Figure (G1G4, H1H4) shows an example of a class IV protein. This class includes 101 proteins that had reduced abundance in biofilms vs planktonic cultures. In biofilm cells the proteins' amounts increased with increasing [Ca2+], but in planktonic cells remained constant. The Class IV protein 12 has strong identity to an intracellular protease of the ThiJ/PfpI family (Glockner et al., 2003
). Protein 16 has similarity to a tetratricopeptide repeat (TPR) protein of I. loihiensis. Class V includes 70 proteins that are of interest since they do not appear to be regulated by the surface, but are influenced by Ca2+. Sequence information was obtained from one of these proteins, which had a low level of similarity to a hypothetical protein from Photorhabdus luminescens.
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DISCUSSION |
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Calcium is thought to allow thicker bacterial biofilms, primarily through ionic bridging of the extracellular matrix material (Korstgens et al., 2001; Rose & Turner, 1998
; Rose, 2000
). Extracellular matrix material is usually composed of negatively charged extracellular polysaccharides, and the cross-linking of these polysaccharides with divalent cations forms a cohesive extracellular gel for biofilms. Calcium-binding proteins may also be important for bacterial adhesion to a surface (Craven & Williams, 1998
; Hinsa et al., 2003
; Matsumoto et al., 2000
; Waligora et al., 1999
). In addition to its role in chemical cross-linker, calcium probably influences regulatory processes in bacteria. Therefore, we tested the responses of Pseudoalteromonas sp. 1398 to differing [Ca2+] while the cells were undergoing a shift from planktonic growth to surface-associated biofilm growth.
Included in the physiological changes that occur in Pseudoalteromonas sp. 1398 in response to both Ca2+, and to a surface, is the production of extracellular matrix material, probably an acidic polysaccharide, which stains with alcian blue. Calcium also influenced extracellular protein production of Pseudoalteromonas sp. 1398. Regulation of extracellular proteins by calcium has been demonstrated for other bacteria, including the myxobacterium Stigmatella aurantiaca (Chang & White, 1992), where the addition of Ca2+ to the growth medium resulted in the formation of extracellular fibrils and the appearance of a 30 kDa fibril protein, and Pseudomonas aeruginosa, where the amounts of certain extracellular proteases and toxins are affected by Ca2+. Our results show that the presence of 10 mM Ca2+ causes changes in extracellular protein biosynthesis and/or accumulation in the marine bacterium Pseudoalteromonas sp. 1398 as well. In particular higher [Ca2+] results in reduced amounts of flagella and flagellin production, while it causes an increase in the amounts of at least four other proteins, including a putative phosphatase. Since these increases occurred under both planktonic and biofilm conditions, it appears that calcium has a greater regulatory influence for these extracellular proteins than does biofilm formation.
The source of the bacterial isolate Pseudoalteromonas sp. 1398 was sea water sediments. The coexistence of environmental factors (elevated [Ca2+] and surface-associated growth) may serve as complementary signals, and cause similar and/or cumulative changes in the bacterial responses. Cluster analysis of protein profiles observed by 2DGE demonstrated that both surface-associated growth and the presence of Ca2+ had a major physiological effect on Pseudoalteromonas sp. 1398, and the presence of both factors had cumulative effects on protein profiles. Following surface-associated growth at 0·25 mM CaCl2, Pseudoalteromonas sp. had 174 protein differences from planktonically grown cultures, including 46 newly appeared proteins. This response represents a change in approximately 22 % of the total number of intracellular proteins resolved by 2DGE. At 10 mM CaCl2, we observed 302 protein differences, representing approximately 38 % of the resolved proteome. This value included 80 protein spots that were only observed in the biofilm culture. Increased [Ca2+] was positively correlated to an increase in the number of newly appearing protein spots. However, the number of proteins showing reduced amounts did not change in response to CaCl2 addition. When the cluster-analysis comparison was performed independently on proteomes of planktonic cultures, or on the proteomes of surface-associated cultures, we found that Ca2+ affects the bacteria differently, with a greater effect seen in the sessile biofilm cultures than in the planktonic cells. The number of proteins showing increased amounts, as well as the number of newly appearing proteins at higher concentrations of calcium, was greater for the biofilm-cultivated cells than for the planktonic bacteria. In addition, there was a positive correlation between the increasing [Ca2+] and number of newly appeared proteins in biofilm cultures, but not planktonic samples. The increase in the number of newly appeared proteins in biofilms compared to planktonic cells could reflect two factors. Firstly, it could be a direct effect of the calcium as a signalling molecule for the biofilm bacteria. Secondly, it could be an indirect effect, due to the increased amount of cell biomass associated with the surface. As the cell density increases, the individual bacteria may experience other environmental variables that could influence gene expression of the surface-associated bacteria. This second scenario, although possible, is less likely in these studies, since the biofilms here were sampled at 7 h, and prior to the development of thick biofilms.
Based on cluster analysis of protein-spot signal intensity, we grouped the proteins of interest into five classes. Fifteen proteins representing different classes were then subjected to sequence analysis by N-terminal peptide sequencing and/or MS/MS analysis. Two proteins, succinyl-CoA synthetase and malate dehydrogenase, were definitively identified as class 1 proteins, which show increased amounts in biofilm culture. Since these proteins are involved in central metabolism, they are not likely to be directly involved in bacterial biofilm formation per se, but rather reflect a change in metabolic activity of the cells as they switch from planktonic to biofilm-associated growth. The proteomics studies performed on Pseudomonas aeruginosa and Escherichia coli biofilms also showed differential amounts of proteins involved in general metabolic activities when comparing planktonic bacteria to biofilm-associated cells (Perrot et al., 2000; Sauer & Camper, 2001
; Tremoulet et al., 2002
). Included in those studies were malate dehydrogenase, thiamine phosphate pyrophosphorylase, aldehyde dehydrogenase, sugar and amino acid transporters and amino acid metabolism enzymes. Other class I proteins shown here have tentative identifications, and include a conserved hypothetical protein related to a Pseudomonas syringae protein, as well as a putative TonB-dependent receptor protein, found in I. loihiensis and in a marine Pirellula sp. These proteins represent novel proteins not identified as being expressed during the biofilm formation of other organisms, and therefore may represent proteins that are specific for biofilm formation by Pseudoalteromonas. Other proteins showing increased amounts in biofilms include two proteins whose identification was not possible. The lack of sequence identity may have been due to the limited sequence information for marine bacteria, or it may reflect amino acid variability in the region of the protein sequenced here.
Class II proteins are of particular interest, since they reflect proteins with increased amounts during calcium-induced biofilm formation. Therefore, these proteins may play a role in the enhanced biofilm thickness that results from calcium addition. As with the class I proteins, a protein involved in central metabolism was identified, PurM. Tentative identifications were made for three other proteins, which include an outer-membrane protein, OmpW, as well as a putative TonB-system biopolymer transport protein. If the biopolymer transport protein is confirmed, it will be consistent with the increased extracellular polysaccharide observed in biofilms with added Ca2+. The other class II proteins could not be positively identified from this amino acid sequence information. Class III and IV proteins included those that had greater expression in planktonic culture. These proteins either are not essential during biofilm growth, or possibly inhibit growth of the bacteria on a surface. One of the class III proteins was sequenced and identified as a probable peroxiredoxin/glutaredoxin protein involved in electron transport. One class IV protein was identified as a putative intracellular protease. The protease had reduced expression in biofilms, and was also down-regulated by Ca2+ addition. The other class IV protein had reduced expression in biofilms, but showed increasing amounts with increased [Ca2+], indicating an inverse response of these proteins to changes in [Ca2+], during surface-associated growth.
Many of the proteins shown here to be differentially expressed are novel and have no homologues with high sequence identity. Therefore, genetic studies will be necessary to determine the role of these proteins in biofilm formation. The results here indicate that the environmental factors, surface-associated growth and calcium addition, cause global changes in the protein profiles of the marine bacterium Pseudoalteromonas sp. 1398. The combination of the two factors shows combinatory effects on the protein profiles. The results also demonstrate that calcium influences Pseudoalteromonas sp. 1398 cells, primarily during biofilm growth.
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ACKNOWLEDGEMENTS |
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Received 16 March 2005;
revised 6 June 2005;
accepted 7 June 2005.
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