Calcium influences cellular and extracellular
product formation during biofilm-associated
growth of a marine Pseudoalteromonas sp.
M. A. Patrauchan,1 S. Sarkisova,2 K. Sauer3 and M. J. Franklin2,4
Correspondence
Michael J. Franklin
umbfm@montana.edu
1Department of Microbiology and Immunology, University of British Columbia, Vancouver,
Canada V6T 1Z3
2,4Department of Microbiology2 and Center for Biofilm Engineering4, Montana State University,
Bozeman, MT 59717, USA
3Department of Biological Sciences, State University of New York at Binghamton,
Binghamton, NY 13902, USA
Received 16 March 2005
Revised 6 June 2005
Accepted 7 June 2005
Bacteria undergo a variety of physiological changes following a switch from planktonic growth
to surface-associated biofilm growth. Here, it is shown that biofilm development of a marine
isolate, Pseudoalteromonas sp. 1398, results in global changes in its cytosolic and extracellular
proteomes. Calcium influences these proteome responses, and affects the amount of
surface-associated biomass and extracellular matrix material produced by Pseudoalteromonas
sp. 1398. Four extracellular proteins, characterized by N-terminal sequencing, showed increased
abundances, while one protein, flagellin, showed reduced abundance at higher [Ca2+].
Immunoblotting and transmission-electron-microscopy analysis confirmed that higher [Ca2+] and
surface-associated growth results in the repression of flagella production. Two-dimensional gel
electrophoresis (2DGE) studies combined with cluster analysis of global proteome responses
demonstrated that Ca2+ had a greater regulatory influence on Pseudoalteromonas sp. growing
in biofilms than on planktonic cultures. Approximately 22% of the total cytosolic proteins
resolved by 2DGE had differing abundances in response to a switch from planktonic growth to
surface-associated growth when the cells were cultivated in 1 mM Ca2+. At higher [Ca2+] this
number increased to 38%. Fifteen cellular proteins that were differentially expressed in response
to biofilm growth and/or Ca2+ were analysed by N-terminal sequencing and/or MS/MS. These
proteins were identified as factors involved in cellular metabolic functions, putative proteases
and transport proteins, although there were several proteins that had not been previously
characterized. These results indicate that Ca2+ causes global changes in matrix material,
as well as in cellular and extracellular protein profiles of Pseudoalteromonas sp. 1398. These
changes are more pronounced when the bacterium grows in biofilms than when it grows in
planktonic culture.
INTRODUCTION
Many bacteria are successful at adapting to a variety of
environmental conditions, in part because they are effec-
tive at sensing environmental stimuli and optimizing gene
expression patterns. This has been demonstrated for bacteria
growing in biofilms, micro-organisms associated with sur-
faces often encased in extracellular matrix material. During
biofilm development, bacteria demonstrate physiological
and sometimes morphological changes, which ultimately
lead to the formation of a mature biofilm (Dalton et al.,
1994; Davies et al., 1993; Sauer et al., 2002; Whiteley et al.,
2001). Proteomic studies have demonstrated differences
in the amounts of as many as 50% of the cellular proteins
during a switch from planktonic growth to biofilm for-
mation and development (Sauer et al., 2002). These physio-
logical changes suggest that bacterial regulatory elements
sense the physical and/or chemical signals associated with
surfaces, and respond to these environmental stimuli
through gene induction and gene repression events.
In marine environments, calcium is an element that is often
Abbreviations: 2DGE, two-dimensional gel electrophoresis; MS/MS,
tandem mass spectrometry; SCLM, scanning confocal laser microscopy;
TEM, transmission electron microscopy.
A figure showing 2DGE of protein extracts is available as supplemen-
tary data with the online version of this paper.
0002-8041 G 2005 SGM Printed in Great Britain 2885
Microbiology (2005), 151, 2885–2897 DOI 10.1099/mic.0.28041-0
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 im-
portant 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 cell–cell 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 concen-
trations may be elevated. Calcium is an important regula-
tory 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 polysaccha-
ride material of an alginate-producing strain of Pseudo-
monas 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, Pseudo-
alteromonas 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 environ-
mental conditions, such as those that would occur during
biofilm formation and development.
Several experimental approaches have been used to charac-
terize 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 sub-
tractive hybridization (Sauer & Camper, 2001), transposon
mutagenesis studies (O’Toole & Kolter, 1998) and proteo-
mic 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 tech-
nologies 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.
METHODS
Organism and growth conditions. The bacterial isolate Pseudo-
alteromonas sp.1398 used in this study was originally isolated from
marine sediments, and was provided by Dr Keith Cooksey (Montana
State University; Wigglesworth-Cooksey & Cooksey, 2005). The
bacterium was isolated by washing sediments extensively to remove
loosely adhered bacteria, and then sonicating to release the tightly
adhered cells. The dislodged bacteria were serially diluted, plated on
2216 marine medium (Difco), and individual colonies isolated. The
isolate used in these studies was identified as a Pseudoalteromonas
sp. by sequence analysis of its 16s rDNA.
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 ml
trace metals solution, 1 ml vitamins solution. Trace metals solu-
tion 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 mediumwas 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 analy-
sis of planktonic cells, bacteria were grown in MMM in culture
2886 Microbiology 151
M. A. Patrauchan and others
flasks (1 : 100 inoculum) with shaking (250 r.p.m.) at room tem-
perature. 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 min21.
For biomass analysis, the mesh was removed from the reactor, and
washed twice inmarine salts buffer (MMMwithout 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 bicin-
choninic 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 30–60 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 micro-
scopy (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 (5126
512 bit resolution) were collected using a Leica TCS NT SCLM
equipped with a 6100 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 6100 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% glutaralde-
hyde, applied to 300 mesh Formvar coated cooper grids, stained
with 1% uranyl acetate and analysed with a Zeiss 100CA transmis-
sion electron microscope.
Extracellular protein analysis. Extracellular proteins were charac-
terized 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 con-
centration) 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 resus-
pended 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 pro-
tein concentrations were determined by a modified BCA method
(Pierce). Protein samples (5 mg) were resolved by 12% SDS-PAGE
(Laemmli, 1970). Proteins were stained with either silver nitrate or
Coomassie blue. Immunoblotting was used for flagellin determina-
tions. 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 200–400 ml TE buffer (10 mM Tris/HCl, 1 mM
EDTA, pH 8?0), containing 0?3 mg PMSF ml21. 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 220 uC 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 4–7, linear; Amersham). The IPG strips were
rehydrated with the protein sample (100–700 mg total protein) in
450 ml rehydration solution (11 M urea, 2?7 M thiourea, 35 mMDTT,
3% CHAPS, pharmalyte pH 3–10). The strips were incubated over-
night at room temperature under mineral oil. After rehydration, IEF
was carried out for a total of 35 kVh at 20 uC, 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 Dyna-
mics) software package. This software detects protein spots, sub-
tracts 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 warp-
ing and matching algorithm. Spots with a normalized volume above
0?02 and averaged over two gels (from independent growth experi-
ments) 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 220 uC. The protein spots of inter-
est 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://mic.sgmjournals.org 2887
Pseudoalteromonas sp. proteomics
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).
RESULTS
Ca2+ does not affect the growth rate of
Pseudoalteromonas sp. in planktonic culture,
but affects biofilm-associated growth on both
hydrophobic and hydrophilic surfaces
Since calcium influences biofilm formation for many
bacteria, we first determined if this effect extended to the
Pseudoalteromonas sp. isolate used here. For these studies,
the bacteria were allowed to attach to and grow on Teflon
mesh or glass surfaces in the presence of differing [Ca2+]
(0?25, 1?0, 5?0 and 10 mM as CaCl2). Cells were then
removed from the surfaces and assayed for total protein as a
measure of the surface-associated biomass. In planktonic
culture, the [Ca2+] in the media had little effect (less than
15%) on the growth rate of Pseudoalteromonas sp. 1398;
the growth curves for each of the culture conditions were
essentially identical (Fig. 1A). However, the addition of
CaCl2 had a significant effect on the biofilm-associated
growth of Pseudoalteromonas sp. 1398 on both hydrophobic
and hydrophilic surfaces (Fig. 1B,C). On glass surfaces,
10 mM CaCl2 resulted in a 20-fold to 100-fold greater
amount of surface-associated biomass compared to biofilm
growth in medium with 0?25 mM CaCl2. Less total biomass
was observed on the Teflon surface compared to the
glass surface (Fig. 1B). However, [Ca2+] affected the total
amount of biomass on this hydrophobic surface as well.
Therefore, although [Ca2+] does not appear to influence
Pseudoalteromonas sp. 1398 growth rate in planktonic cul-
ture, it significantly impacts its ability to colonize both
hydrophobic and hydrophilic surfaces.
Scanning confocal laser microscopy (SCLM) was used as
a second method to determine the effect of Ca2+ on Pseudo-
alteromonas sp. biofilm formation. Fig. 2 (A–D) shows
SCLM images of a Pseudoalteromonas sp. biofilm after 30 h
growth when the [Ca2+] was varied from 0?25 to 10 mM.
The BacLight staining of the biofilms did not show signi-
ficant amounts of nonviable cells associated with biofilms.
At low [Ca2+] the surface contained single cells and small
groups of cells with few bacteria extending more than one
cell deep from the surface. In contrast, the presence of 1 and
5 mM CaCl2 resulted in the formation of cell clusters that
extended 20 to 40 mm from the surface. Thicker biofilms
(approximately 50 mm) formed in the presence of 10 mM
CaCl2. These experiments demonstrated that the increased
biomass associated with the surface observed with the total
protein analyses was due to the formation of thicker micro-
colonies that extend from the solid substratum.
Ca2+ influences the amount of extracellular
matrix material associated with the biofilm
In a mucoid strain of Pseudomonas aeruginosa, Ca2+ caused
increased production of the exopolysaccharide alginate
during biofilm formation (Sarkisova et al., 2005). Since
Ca2+ also affects the biomass and thickness of the biofilms
of this marine Pseudoalteromonas sp., it is possible that
this increased thickness is due to an increased amount of
extracellular polysaccharide matrix material produced by
the bacteria. To test this hypothesis, we performed alcian
blue staining of the biofilms at various [Ca2+]. The results
in Fig. 2 (E–H) show increased amounts of stained mater-
ial surrounding the cell clusters with increased [Ca2+].
Since alcian blue stains acidic polysaccharides, the results
1.6
1.2
0.8
0.4
4 8 12 16 20 24 28 32 36 40
16
12
8
4
5
4
3
2
1
31 43 79 103
7 19 31 43
0.25 mM CaCl2
1.0 mM
5.0 mM
10.0 mM
(A)
(B)
(C)
Time (h)
Fig. 1. Growth curves of Pseudoalteromonas sp. 1398 in mini-
mal medium at differing [CaCl2]. (A) Growth in planktonic
culture for cells in 0?25, 1?0, 5?0 and 10?0 mM CaCl2. Total
cellular protein of Pseudoalteromonas sp. 1398 biofilms asso-
ciated with (B) glass surfaces and (C) Teflon surfaces. Biofilms
were cultured in MMM with 0?25, 1?0, 5?0 and 10?0 mM
added CaCl2.
2888 Microbiology 151
M. A. Patrauchan and others
indicate that this Pseudoalteromonas sp. produces
larger amounts of biofilm-associated polysaccharide with
increased [Ca2+].
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).
Fig. 2. SCLM images of Pseudoalteromonas sp. 1398 biofilms, cultivated for 30 h on glass surfaces at different [CaCl2]: (A)
0?25 mM, (B) 1?0 mM, (C) 5?0 mM and (D) 10?0 mM CaCl2. Alcian blue stained Pseudoalteromonas sp. 1398 biofilms
cultivated at different [CaCl2]: (E) 0?25 mM, (F) 1?0 mM, (G) 5?0 mM and (H) 10?0 mM CaCl2.
Fig. 3. TEM images of Pseudoalteromonas
sp. 1398. (A) Cells grown in planktonic cul-
ture with 0?25 mM CaCl2, showing polar fla-
gella. (B) Cells grown in planktonic culture
with 10?0 mM CaCl2, showing production of
intercellular matrix material but no flagella.
(C) Cells from a biofilm cultivated in
0?25 mM CaCl2, showing some matrix mater-
ial, but no flagella. (D) Cells from a biofilm
cultivated with 10?0 mM CaCl2, showing an
increased amount of matrix material.
http://mic.sgmjournals.org 2889
Pseudoalteromonas sp. proteomics
Ca2+ affects extracellular proteins produced by
Pseudoalteromonas sp. 1398
The TEM results for the planktonic cultures indicated that
Ca2+ affects the production of flagella by this species of
Pseudoalteromonas. Since Ca2+ influences several secreted
factors of Pseudomonas aeruginosa, it is possible that it
also influences the production of extracellular proteins
of Pseudoalteromonas sp. To test this, we extracted, TCA
precipitated and analysed extracellular proteins from both
biofilm and planktonic cultures incubated at differing
[Ca2+]. In planktonic cultures at low [Ca2+] one dominant
protein band was observed (Fig. 4A, protein 1). This band
was blotted onto PVDF membrane, excised and analysed by
N-terminal sequence analysis. The N-terminal sequence of
this protein had 93% identity to the flagellin protein, FlaA,
of Aeromonas punctata. Identification of flagellin under
planktonic conditions at low concentration of Ca2+ is con-
sistent with the TEM results showing single polar flagella
in these cells. To verify that this protein was flagellin, we
immunoblotted the TCA-precipitated proteins from biofilm
cultures and from planktonic cultures, using an antibody
developed against the flagellin of Pseudomonas aeruginosa
(PAK and PAO1). The results in Fig. 4(C) show a band
corresponding to flagellin under all conditions tested:
planktonic growth at 0?25 and 10?0 mM Ca2+, and biofilm
growth at 0?25 and 10 mM Ca2+. However, in agreement
with the TEM results, the greatest intensity of the flagellin
band was observed in samples from the planktonic culture at
low levels of Ca2+. The least intense band was observed in
the sample from the biofilm culture at 10 mM Ca2+. The
flagellin observed in the biofilm cultures was likely to be due
to the flagellin debris, as was observed by TEM.
When the [Ca2+] was increased to 10 mM, at least four
extracellular proteins showed increased amounts in plank-
tonic culture, with the flagellin band showing reduced
amounts (Fig. 4A). In biofilm at low [Ca2+], many bands
were observed that may have been derived from debris from
lysed cells. However, at increased [Ca2+] the four bands that
were observed in planktonic culture at high [Ca2+] were
also observed in biofilms (Fig. 4B). These proteins were not
observed in biofilm culture at lower [Ca2+], indicating that
Ca2+ has a greater regulatory influence on extracellular
protein production in Pseudoalteromonas sp. than does
biofilm-associated growth. The four extracellular proteins
that showed increased expression with Ca2+ addition were
characterized by either N-terminal sequence analysis or MS/
MS sequencing of peptide fragments. The protein labelled 2
(the 71 kDa protein) had a repeating GADGVD sequence,
also found in secreted proteins from ‘Microbulbifer degra-
dans’, Corynebacterium glutamicum and Bacteroides fragilis.
In ‘M. degradans’, the protein showing sequence similarity is
predicted to be a secreted phosphatase. The sequence iden-
tity for protein 3 (the 64 kDa protein) was too low tomake a
definitive identification. Protein 4 (the 106 kDa protein)
had low levels of sequence similarity to a conserved hypo-
thetical protein from Clostridium thermocellum. Protein 5
Planktonic
0.25 10.0 0.25 10.0
(A) (B)
(C)
4
5
2
3
4
5
2
3
Biofilm
Flagellin
[CaCl2] (mM):
1
Fig. 4. Extracellular proteins produced by Pseudoalteromonas
sp. 1398 during growth in MMM supplemented with 0?25 or
10?0 mM CaCl2. (A) Growth in planktonic culture. Proteins were
resolved by SDS-PAGE and stained with Coomassie blue. (B)
Growth in biofilm culture. The results of N-terminal and MS/MS
sequence analysis of proteins 1–5 are shown in Table 1. (C)
Immunoblot analysis of cells cultivated using the same conditions
as for the Coomassie blue-stained gels, with antibodies raised
against flagellin from Pseudomonas aeruginosa.
Table 1. N-terminal and MS/MS analysis of extracellular
proteins produced by Pseudoalteromonas sp. 1398
Protein Peptide sequence Protein with highest
similarity*
1D ALYVNTNVSALNAQR Flagellin (Aeromonas
punctata) 93%
2d GVDGADGADGVDG Predicted phosphatase
(‘M. degradans’) 92%
3d N-terminal analysis
GVLPDNQMKSDXYSA Unidentified protein
MS/MS analysis
HYTLGR
AAFLESR
RNFDNLPR
FQGDEQVSR
PVLFGGTYR
YSSQDDTGGVLFK
LNSPVPESEFER
ADQPEDLSAGTLYQK
SDVLANEVVTK
4d ALLIDEDVEEGEF Conserved uncharacterized
protein
5d SSDDDDKIESLTVKNT Unidentified protein
*Sequence identities are based on the closest homologues identified
using BLAST searches as described in Methods. Percentage identity of
the peptide to the protein from the organism is indicated.
DProtein up-regulated by low [CaCl2].
dProtein up-regulated by high [CaCl2].
2890 Microbiology 151
M. A. Patrauchan and others
(the 85 kDa protein) could not be identified because of its
lack of sequence similarity to other known proteins.
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 Pseudo-
alteromonas 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 con-
dition (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 bio-
film 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.
To determine the role of Ca2+ in the proteomic response of
Pseudoalteromonas sp. 1398 in both planktonic and biofilm
culture, we compared protein profiles of cells incubated at
0?25 and 10?0 mM [Ca2+]. Fig. 6 shows the ratios of log10
values of the signal intensities for each protein spot for the
planktonic (Fig. 6A) and biofilm cells (Fig. 6B). The data
(A) (B)
0.01 0.1 1
0.01
0.1
1
Relative protein amount (planktonic)
R
el
at
iv
e 
pr
ot
ei
n 
am
ou
nt
 (b
io
fil
m
)
Fig. 5. (A) 2DGE analysis of Pseudoalteromonas sp. 1398 cellular proteins. Cells were cultured in a biofilm for 7 h in a once
flow-through reactor with minimal medium and 1?0 mM CaCl2. Proteins were resolved using pH 4–7 isoelectric focusing and
10% SDS-PAGE. Highlighted are protein spots showing different amounts between planktonic cells and surface-associated
cells, as determined by Progenesis software for two or more independent experiments. Shown in grey are spots that have
twofold or greater increased intensity in biofilm cultures versus planktonic cells. Shown in black are protein spots that were
only observed in biofilm cultures, with no corresponding spot in planktonic cultures. Shown in white are spots with twofold or
greater increased amounts in planktonic cells versus biofilm cultures. (B) Analysis of signal intensity for each protein resolved
by 2DGE. Spot signal intensity from biofilm culture (y-axis) plotted against its intensity from planktonic culture (x-axis). Each
point represents the mean intensity for a protein from two or more gels under each growth condition. Proteins represented
above the top line are those with twofold or greater intensity in biofilm culture. Proteins represented below the bottom line are
those with twofold or greater intensity in planktonic culture. Proteins represented along the y- and x-axes are those only
observed in biofilm or planktonic culture, respectively.
http://mic.sgmjournals.org 2891
Pseudoalteromonas sp. proteomics
distribution had a greater range (a greater scatter of the
protein-spot signal intensities) for the biofilm bacteria than
for the planktonic cells, indicating that the increased [Ca2+]
has less effect on the bacteria grown planktonically, than on
biofilm-associated cells. For the planktonic cells, most of the
protein spot intensities lie within the twofold range, show-
ing equivalent amounts of protein under both conditions
(0?25 and 10 mM CaCl2), with 61 proteins having at least a
twofold greater intensity in the 10 mM CaCl2 medium, and
26 proteins only found when the cells were cultured with
10 mM CaCl2 (spots along the y-axis). In contrast, the
proteomes had a greater number of proteins that were
outside of the twofold range for the biofilm cultures cul-
tivated at differing [Ca2+] (Fig. 6B). In this case, 159 pro-
teins had at least a twofold increase in intensity in the
10 mM CaCl2 medium. Of these, 88 proteins were found
only under the 10 mM [Ca2+] condition. The results indi-
cate that Ca2+ has a regulatory effect (or protein stability
effect) on many proteins during planktonic growth. How-
ever, [Ca2+] exerts a much more pronounced effect on the
proteome of biofilm-associated cells.
Experiments where the bacteria were shifted from plank-
tonic growth to biofilm-associated growth were performed
at four [Ca2+] (0?25, 1?0, 5?0 and 10 mM). The results in
Table 2 show the cluster-analysis results of proteins with
twofold or greater increased or decreased abundance in
biofilm culture versus planktonic culture for each [Ca2+].
The results demonstrate that the number of protein spots
showing increased intensity in biofilms compared to plank-
tonic cultures increases with increasing [Ca2+]. For example,
at 0?25 mM [Ca2+], 174 proteins show changed abundance
in biofilm vs planktonic cultures, with 76 showing at least
a twofold increase in abundance, 46 appearing only in the
biofilm cultures and 52 showing decreased abundance. In
contrast, at 10 mM CaCl2 the number of proteins showing
increased abundance almost doubled, with 167 showing a
twofold or greater increased abundance in biofilm culture,
80 proteins only appearing in biofilm culture, and 55 show-
ing decreased abundance in biofilm culture. These results
demonstrate that Pseudoalteromonas has a greater regula-
tory response to a shift from planktonic culture to biofilm
culture as the concentration of Ca2+ increases.
Cluster analysis and identification of proteins
showing differing expression patterns
We grouped the cytosolic proteins into five classes based on
the conditions under which they were differentially expres-
sed (for the four different [Ca2+], and with at least two
experiments per [Ca2+]). Class I proteins (27 proteins)
showed increased abundance in biofilm cells, but were
unaffected by Ca2+. An example of a class I protein (spot 1)
is shown in the Supplementary Figure (A1–A4, B1–B4
(A) (B)
0.01 0.1 1
0.01
0.1
1
Relative protein amount (0.25 mM CaCl2)
0.01 0.1 1
0.01
0.1
1
Fig. 6. Cluster analysis of the effect of Ca2+ on global protein profiles of planktonic and surface-associated Pseudo-
alteromonas sp. 1398. (A) Individual protein-spot signal intensity for planktonic cultures plotted for cultures incubated in
minimal medium with 10 mM CaCl2 (y-axis) versus spot intensity in planktonic culture at 0?25 mM CaCl2 (x-axis). Spots
above the top line and below the bottom line indicate proteins with twofold or greater amounts in 10 and 0?25 mM CaCl2,
respectively. Spots along the y-axis are proteins only found when cells are cultivated in 10 mM CaCl2, and those along the
x-axis are those that are only found when cells are grown in 0?25 mM CaCl2 medium. (B) Cluster analysis of individual protein
spots for cells cultivated in biofilms, when cells are exposed to 10 (y-axis) and 0?25 (x-axis) mM CaCl2. All other parameters
are the same as in (A). Data represent the mean of intensities from duplicate independent experiments.
2892 Microbiology 151
M. A. Patrauchan and others
available with the online journal). N-terminal sequence and/
or MS/MS analysis were used to identify peptide sequences
for six class I proteins (Table 3). For protein spot 1 both N-
terminal sequencing and MS/MS gave identical sequences
for the N-terminus of the protein, verifying that both
techniques were useful for obtaining peptide sequences.
Based on the sequence identity, and consistent with the
predicted pI and molecular mass, the protein’s most likely
homologue is a hypothetical protein from Pseudomonas
syringae. Protein spot 2 has the highest sequence identity to
a hypothetical protein from a marine Pirellula sp. (Glockner
et al., 2003), and to a putative TonB-dependent receptor
from Sinorhizobium meliloti (Finan et al., 2001) and Idio-
marina loihiensis (Hou et al., 2004). Class I proteins spots 9
and 13 could not be identified due to inadequate amino acid
sequence identity to known proteins. Protein spots 6 and 8
were identified as the metabolic proteins malate dehydro-
genase and succinyl-CoA synthetase.
Class II included 39 proteins that showed increased abun-
dance in biofilms, and were also influenced by Ca2+
(Supplementary Figure, C1–C4, D1–D4, shows a represen-
tative class II protein- spot 3). These proteins were not
found in planktonic cultures at any [Ca2+], but occurred in
biofilm cells at low abundance and showed the greatest
abundance in biofilms at 10 mM CaCl2. The peptide
sequences of six class II proteins are shown in Table 3.
Sequence homology searches revealed strong identity for
protein 10, identified as PurM. Other proteins also showed
identity to known proteins, including protein 5, which has
similarity to a TonB-dependent biopolymer transport
protein of I. loihiensis (Hou et al., 2004), protein 3, which
has similarity to the outer-membrane protein OmpW of
I. loihiensis (Hou et al., 2004), and protein 7, which has
similarity to a conserved hypothetical protein. Protein 1270
did not have strong identity to any known proteins.
Twenty-four proteins were grouped as class III, those with
decreased intensity in biofilms, but unaffected by Ca2+
(example shown in the Supplementary Figure E1–E4 and
F1–F4, labelled spots 14 and 15). N-terminal peptide
sequences of protein 14, gave strong identity to a pro-
tein in peroxiredoxin/glutaredoxin family (Heidelberg
et al., 2000). The Supplementary Figure (G1–G4, H1–H4)
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.
DISCUSSION
When bacteria adhere to surfaces, they sense chemical or
physical properties associated with the surface, and undergo
physiological changes. These changes include expression of
genes that allow them to firmly adhere to the surface, termed
secondary adhesion, and ultimately to develop into struc-
tured biofilms. Genes expressed and proteins produced dur-
ing biofilm formation have been characterized for several
organisms (Kachlany et al., 2001; Kuchma & O’Toole, 2000;
Perrot et al., 2000; Pratt & Kolter, 1999; Ren et al., 2004;
Sauer et al., 2002; Schoolnik et al., 2001; Tremoulet et al.,
2002; Whiteley et al., 2001; Zhu & Mekalanos, 2003). Here,
we report on the global changes in physiology during the
initial stages of biofilm formation of a marine bacterium,
Pseudoalteromonas sp. 1398. These studies provide informa-
tion on the physiological response of a marine organism to a
shift from planktonic to sessile biofilm growth, and there-
fore can be used to characterize similarities between biofilm
formation in this marine isolate and that in freshwater
isolates, as well as bacteria of medical importance. In addi-
tion, this study provides information on global changes
in protein profiles resulting from a combination of two
environmental parameters, surface-associated growth and
the concentration of calcium in the medium. Calcium con-
centration was chosen as an environmental variable since
it is known to influence the biofilm formation of certain
bacteria (Geesey et al., 2000). Here we show that Ca2+
also influences the formation of biofilms by this Pseudo-
alteromonas isolate.
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
Table 2. Comparison of the no. of protein differences
between surface-associated biofilm cells and planktonic
bacteria at differing [Ca2+]
Concn
CaCl2
(mM)
Protein differences
No. increased
in biofilm*
No. newly
appeared
in biofilmD
No. decreased
in biofilmd
Total
0?25 76 46 52 174
1?0 93 62 47 202
5?0 108 74 45 227
10?0 167 80 55 302
*Proteins present under both biofilm and planktonic conditions, but
with at least twofold increased amounts in biofilms for two or more
independent experiments.
DProteins only found in biofilm-cultivated cells and not present in
planktonic cultures.
dProteins with at least a twofold increased amount in extracts from
planktonic cultures compared to extracts of biofilm-cultivated cells.
http://mic.sgmjournals.org 2893
Pseudoalteromonas sp. proteomics
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 prob-
ably 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 Pseudo-
alteromonas 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 produc-
tion of Pseudoalteromonas sp. 1398. Regulation of extra-
cellular proteins by calcium has been demonstrated for other
bacteria, including the myxobacterium Stigmatella auran-
tiaca (Chang & White, 1992), where the addition of Ca2+
to the growth medium resulted in the formation of extra-
cellular fibrils and the appearance of a 30 kDa fibril pro-
tein, 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+
Table 3. Peptide sequences of biofilm and/or calcium-regulated proteins
Class Protein Method* Peptide sequence kDaD pId Protein with highest similarity§
I 1 N-terminal SNETYTFNAELRTATG 23 5?8 Conserved hypothetical protein (Pseudomonas syringae), or
ribosomal protein (Pseudomonas putida, 67%)MS/MS SNETYTFNAELR
LPGAVSSVDLAK
VVHFLGEEAVTK
VPVHFLDAEAVTK
LDFQR
LTHLQFDR
PAGVSSVDLAK
ASLTEFHK
2 N-terminal ETQELLAEYRSVVDE 23 5?2 Putative TonB-dependant receptor (I. loihiensis, 67%)
6 N-terminal SVLINKDTKVIXQGF 30 5?5 Succinyl-CoA synthetase (P. fluorescens, 100%)
8 N-terminal MKVAVLGAAGGIGQA 31 4?8 Malate dehydrogenase (I. loihiensis, 100%)
MS/MS AEGLMTLVPK
WLFNVNAGLLK
SEAFVAELK
FCMSLVK
LQNAEGTEVVQGK
VFGLTTDVLR
9 N-terminal ATIGGTDVTYGGYIK 34 4?8 No identification
13 N-terminal AEQQVQPLHNEKHAN 26 5?8 No identification
II 5 MS/MS NVDFGQLGR 24 5?2 TonB-system biopolymer transport component (I. loihiensis)
FLDTLEQFLK
LTYVAQSFDLK
LVADQNASLESFDR
AGLQLQTAAVK
VDESELLK
DAVPFNSDNR
3 N-terminal NLSVNVGAINVNPDN 20 4?2 OmpW (I. loihiensis, 60%)
10 N-terminal SEQKQSLSYKDAGVD 34 4?9 PurM (95%)
7 N-terminal AEYDIYGKAEVQIAN 27 5?5 Conserved protein (Burkholderia fungorum, 67%)
1270 MS/MS LLGTETFR 52 5?7 No identification
AEPSQLA
III 14 N-terminal MLKNIEGQTIPQVTF 26 5?0 Peroxiredoxin/glutaredoxin family protein (V. cholerae, 67%)
IV 12 N-terminal AKVLMITGDFVEDYE 34 5?5 ThiJ/PfpI family of proteases (D. vulgaris, 86%)
16 EEVKTKRVPALREKV 26 5?0 TPR repeats protein (I. loihiensis, 67%)
V c N-terminal GVLPDNQMKSDWYSA 51 4?6 Hypothetical protein Photorhabdus luminescens
*Peptide sequences were obtained using either N-terminal or MS/MS analysis as described in Methods.
DEstimated molecular mass of the proteins, based on their positions in the SDS-PAGE gels.
dEstimated charge of the proteins based on their positions on the IPG strips.
§Sequence identities are based on the closest homologues identified using BLAST searches as described in Methods.
2894 Microbiology 151
M. A. Patrauchan and others
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 environ-
mental factors (elevated [Ca2+] and surface-associated
growth) may serve as complementary signals, and cause
similar and/or cumulative changes in the bacterial res-
ponses. 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 approxi-
mately 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 pro-
teomes 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 plank-
tonic samples. The increase in the number of newly app-
eared 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 meta-
bolism, 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 plank-
tonic 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, thi-
amine phosphate pyrophosphorylase, aldehyde dehydro-
genase, sugar and amino acid transporters and amino acid
metabolism enzymes. Other class I proteins shown here have
tentative identifications, and include a conserved hypothe-
tical 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 there-
fore 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 identi-
fications 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 bio-
polymer 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 expres-
sion in biofilms, and was also down-regulated by Ca2+
addition. The other class IV protein had reduced expression
http://mic.sgmjournals.org 2895
Pseudoalteromonas sp. proteomics
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 expres-
sed 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 bac-
terium Pseudoalteromonas sp. 1398. The combination of the
two factors shows combinatory effects on the protein pro-
files. The results also demonstrate that calcium influences
Pseudoalteromonas sp. 1398 cells, primarily during biofilm
growth.
ACKNOWLEDGEMENTS
We thank Dr Keith Cooksey from for providing the Pseudoalteromonas
sp. 1398 strain.We thankDrDanielWozniak for providing the flagellin
antibodies.We thankDr L. Eltis for support of theMS/MS analyses.We
thank David McCourt for the N-terminal sequence analysis. This work
was supported by a grant from the Office of Naval Research: N00014-
99-1-0678.
REFERENCES
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z.,
Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res 25,
3389–3402.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman,
J. G. & Struhl, K. (1988). Current Protocols in Molecular Biology.
Canada: Wiley.
Chang, B. Y. & White, D. (1992). Cell surface modifications induced
by calcium ion in the myxobacterium Stigmatella aurantiaca.
J Bacteriol 174, 5780–5787.
Craven, S. E. & Williams, D. D. (1998). In vitro attachment of
Salmonella typhimurium to chicken cecal mucus: effect of cations and
pretreatment with Lactobacillus spp. isolated from the intestinal
tracts of chickens. J Food Prot 61, 265–271.
Dalton, H. M., Poulsen, L. K., Halasz, P., Angles, M. L., Goodman,
A. E. & Marshall, K. C. (1994). Substratum-induced morphological
changes in a marine bacterium and their relevance to biofilm
structure. J Bacteriol 176, 6900–6906.
Davies, D. G., Chakrabarty, A. M. & Geesey, G. G. (1993). Exopoly-
saccharide production in biofilms: substratum activation of alginate
gene expression by Pseudomonas aeruginosa. Appl Environ Microbiol
59, 1181–1186.
Egan, S., Thomas, T., Holmstrom, C. & Kjelleberg, S. (2000).
Phylogenetic relationship and antifouling activity of bacterial
epiphytes from the marine alga Ulva lactuca. Environ Microbiol 2,
343–347.
Espinosa-Urgel, M., Salido, A. & Ramos, J. L. (2000). Genetic
analysis of functions involved in adhesion of Pseudomonas putida
to seeds. J Bacteriol 182, 2363–2369.
Finan, T. M., Weidner, S., Wong, K. & 8 other authors (2001). The
complete sequence of the 1,683-kb pSymB megaplasmid from the
N2-fixing endosymbiont Sinorhizobium meliloti. Proc Natl Acad Sci
U S A 98, 9889–9894.
Finelli, A., Gallant, C. V., Jarvi, K. & Burrows, L. L. (2003). Use of
in-biofilm expression technology to identify genes involved in
Pseudomonas aeruginosa biofilm development. J Bacteriol 185,
2700–2710.
Geesey, G. G., Wigglesworth-Cooksey, B. & Cooksey, K. E. (2000).
Influence of calcium and other cations on surface adhesion of
bacteria and diatoms: a review. Biofouling 15, 195–205.
Gerhardt, P. E. (1994). Methods for General and Molecular
Bacteriology. Washington, DC: American Society for Microbiology.
Glockner, F. O., Kube, M., Bauer, M. & 11 other authors (2003).
Complete genome sequence of the marine planctomycete Pirellula
sp. strain 1. Proc Natl Acad Sci U S A 100, 8298–8303.
Gorg, A. (1999). IPG-Dalt of very alkaline proteins. Methods Mol Biol
112, 197–209.
Gorg, A., Boguth, G., Obermaier, C., Posch, A. & Weiss, W. (1995).
Two-dimensional polyacrylamide gel electrophoresis with immobi-
lized pH gradients in the first dimension (IPG-Dalt): the state of
the art and the controversy of vertical versus horizontal systems.
Electrophoresis 16, 1079–1086.
Gorg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B.,
Wildgruber, R. & Weiss, W. (2000). The current state of two-
dimensional electrophoresis with immobilized pH gradients.
Electrophoresis 21, 1037–1053.
Heidelberg, J. F., Eisen, J. A., Nelson, W. C. & 23 other authors
(2000). DNA sequence of both chromosomes of the cholera
pathogen Vibrio cholerae. Nature 406, 477–483.
Hinsa, S. M., Espinosa-Urgel, M., Ramos, J. L. & O’Toole, G. A.
(2003). Transition from reversible to irreversible attachment during
biofilm formation by Pseudomonas fluorescens WCS365 requires an
ABC transporter and a large secreted protein. Mol Microbiol 49,
905–918.
Holland, I. B., Jones, H. E., Campbell, A. K. & Jacq, A. (1999). An
assessment of the role of intracellular free Ca2+ in E. coli. Biochimie
81, 901–907.
Holmstrom, C. & Kjelleberg, S. (1999). Marine Pseudoalteromonas
species are associated with higher organisms and produce bio-
logically active extracellular agents. FEMS Microbiol Ecol 30,
285–293.
Hou, S. J. H., Saw, K. S., Lee, T. & 19 other authors (2004). Genome
sequence of the deep-sea gamma-proteobacterium Idiomarina
loihiensis reveals amino acid fermentation as a source of carbon
and energy. Proc Natl Acad Sci U S A 101, 18036–18041.
Kachlany, S. C., Planet, P. J., DeSalle, R., Fine, D. H. & Figurski,
D. H. (2001). Genes for tight adherence of Actinobacillus actinomy-
cetemcomitans: from plaque to plague to pond scum. Trends
Microbiol 9, 429–437.
Kallstrom, H., Hansson-Palo, P. & Jonsson, A. B. (2000). Cholera
toxin and extracellular Ca2+ induce adherence of non-piliated
Neisseria: evidence for an important role of G-proteins and Rho in
the bacteria-cell interaction. Cell Microbiol 2, 341–351.
Kierek, K. & Watnick, P. I. (2003). The Vibrio cholerae O139 O-antigen
polysaccharide is essential for Ca2+-dependent biofilm development in
sea water. Proc Natl Acad Sci U S A 100, 14357–14362.
Korstgens, V., Flemming, H. C., Wingender, J. & Borchard, W.
(2001). Influence of calcium ions on the mechanical properties of a
model biofilm of mucoid Pseudomonas aeruginosa. Water Sci Technol
43, 49–57.
Kuchma, S. L. & O’Toole, G. A. (2000). Surface-induced and biofilm-
induced changes in gene expression. Curr Opin Biotechnol 11,
429–433.
Laemmli, U. K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
2896 Microbiology 151
M. A. Patrauchan and others
Matsumoto, M., Yoshida, E., Takeyama, H. & Matsunaga, T. (2000).
Floating cultivation of marine cyanobacteria using coal fly ash. Appl
Biochem Biotechnol 84–86, 51–57.
Michiels, J., Xi, C., Verhaert, J. & Vanderleyden, J. (2002). The
functions of Ca2+ in bacteria: a role for EF-hand proteins? Trends
Microbiol 10, 87–93.
Nivens, D. E., Ohman, D. E., Williams, J. & Franklin, M. J. (2001).
Role of alginate and its O acetylation in formation of Pseudomonas
aeruginosa microcolonies and biofilms. J Bacteriol 183, 1047–1057.
Norris, V., Grant, S., Freestone, P., Canvin, J., Sheikh, F. N., Toth, I.,
Trinei, M., Modha, K. & Norman, R. I. (1996). Calcium signalling in
bacteria. J Bacteriol 178, 3677–3682.
O’Toole, G. A. & Kolter, R. (1998). Initiation of biofilm formation in
Pseudomonas fluorescens WCS365 proceeds via multiple, convergent
signalling pathways: a genetic analysis. Mol Microbiol 28, 449–461.
Perrot, F., Hebraud, M., Charlionet, R., Junter, G. A. & Jouenne, T.
(2000). Protein patterns of gel-entrapped Escherichia coli cells differ
from those of free-floating organisms. Electrophoresis 21, 645–653.
Pratt, L. A. & Kolter, R. (1999). Genetic analyses of bacterial biofilm
formation. Curr Opin Microbiol 2, 598–603.
Rabilloud, T. (2000). Detecting proteins separated by 2-D gel
electrophoresis. Anal Chem 72, 48A–55A.
Ren, D., Bedzyk, L. A., Thomas, S. M., Ye, R. W. & Wood, T. K.
(2004). Gene expression in Escherichia coli biofilms. Appl Microbiol
Biotechnol 64, 515–524.
Rose, R. K. (2000). The role of calcium in oral streptococcal
aggregation and the implications for biofilm formation and
retention. Biochim Biophys Acta 1475, 76–82.
Rose, R. K. & Turner, S. J. (1998). Extracellular volume in
streptococcal model biofilms: effects of pH, calcium and fluoride.
Biochim Biophys Acta 1379, 185–190.
Sarkisova, S., Patrauchan, M. A., Berglund, D., Nivens, D. E. &
Franklin, M. J. (2005). Calcium-induced virulence factors associated
with the extracellular matrix of mucoid Pseudomonas aeruginosa
biofilms. J Bacteriol 187, 4327–4337.
Sauer, K. & Camper, A. K. (2001). Characterization of phenotypic
changes in Pseudomonas putida in response to surface-associated
growth. J Bacteriol 183, 6579–6589.
Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W. & Davies,
D. G. (2002). Pseudomonas aeruginosa displays multiple phenotypes
during development as a biofilm. J Bacteriol 184, 1140–1154.
Schoolnik, G. K., Voskuil, M. I., Schnappinger, D., Yildiz, F. H., Meibom,
K., Dolganov, N. A., Wilson, M. A. & Chong, K. H. (2001).Whole genome
DNA microarray expression analysis of biofilm development by Vibrio
cholerae O1 E1 Tor. Methods Enzymol 336, 3–18.
Shevchenko, A., Sunyaev, S., Loboda, A., Bork, P., Ens, W. & Standing,
K. G. (2001). Charting the proteomes of organisms with unsequenced
genomes by MALDI-quadrupole time-of-flight mass spectrometry and
BLAST homology searching. Anal Chem 73, 1917–1926.
Skovhus, T. L., Ramsing, N. B., Holmstrom, C., Kjelleberg, S. &
Dahllof, I. (2004). Real-time quantitative PCR for assessment of
abundance of Pseudoalteromonas species in marine samples. Appl
Environ Microbiol 70, 2373–2382.
Tremoulet, F., Duche, O., Namane, A., Martinie, B. & Labadie, J. C.
(2002). A proteomic study of Escherichia coli O157 :H7 NCTC 12900
cultivated in biofilm or in planktonic growth mode. FEMS Microbiol
Lett 215, 7–14.
Waligora, A. J., Barc, M. C., Bourlioux, P., Collignon, A. & Karjalainen,
T. (1999). Clostridium difficile cell attachment is modified by environ-
mental factors. Appl Environ Microbiol 65, 4234–4238.
Whiteley, M., Bangera, M. G., Bumgarner, R. E., Parsek, M. R.,
Teitzel, G. M., Lory, S. & Greenberg, E. P. (2001). Gene expression in
Pseudomonas aeruginosa biofilms. Nature 413, 860–864.
Wigglesworth-Cooksey, B. & Cooksey, K. E. (2005). Use of
fluorophore-conjugated lectins to study cell-cell interactions in
model marine biofilms. Appl Environ Microbiol 71, 428–435.
Yang, K. (2001). Prokaryotic calmodulins: recent developments and
evolutionary implications. J Mol Microbiol Biotechnol 3, 457–459.
Zhu, J. & Mekalanos, J. J. (2003). Quorum sensing-dependent bio-
films enhance colonization in Vibrio cholerae. Dev Cell 5, 647–656.
http://mic.sgmjournals.org 2897
Pseudoalteromonas sp. proteomics