Proteomics dedicated to biofilmology: What have we learned from a decade of research?

Med Microbiol Immunol DOI 10.1007/s00430-015-0423-0

REVIEW

Proteomics dedicated to biofilmology: What have we learned from a decade of research? Arbia Khemiri1,2,3 · Thierry Jouenne1,2,3 · Pascal Cosette1,2,3

Received: 3 September 2014 / Accepted: 3 June 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract Advances in proteomics techniques over the past decade, closely integrated with genomic and physicochemical approach, have played a great role in developing knowledge of the biofilm lifestyle of bacteria. Despite bacterial proteome versatility, many studies have demonstrated the ability of proteomics approaches to elucidating the biofilm phenotype. Though these investigations have been largely used for biofilm studies in the last decades, they represent, however, a very low percentage of proteomics works performed up to now. Such approaches have offered new targets for combating microbial biofilms by providing a comprehensive quantitative and qualitative overview of their protein cell content. Herein, we summarized the state of the art in knowledge about biofilm physiology after one decade of proteomic analysis. In a second part, we highlighted missing research tracks for the next decade, emphasizing the emergence of posttranslational modifications in proteomic studies stemming from recent advances in mass spectrometry-based proteomics. Keywords Biofilm · Proteomics · Subcellular proteomics · Signaling · Posttranslational modifications Abbreviations MS Mass spectrometry

* Arbia Khemiri [email protected] 1

CNRS, UMR 6270, Laboratory “Polymères, Biopolymères, Surfaces”, 76820 Mont‑Saint‑Aignan, France

2

University of Normandy, UR, Mont‑Saint‑Aignan, France

3

PISSARO Proteomic Facility, IRIB, 76820 Mont‑Saint‑Aignan, France

2D-E Two-dimensional electrophoresis CF Cystic fibrosis PTMs Posttranslational modifications EPS Extracellular polymeric substances OMVs Outer membrane vesicles QS Quorum sensing AHLs Acyl homoserine lactones OMP Outer membrane protein

Introduction Every year, the damages caused by bacterial biofilms cost world economy several billions of euros. These damages concern human health [1–4]; industries, including the food industry [5–7]; agriculture [8]; shipping [9]; and water quality [10, 11]. In the meantime, biofilms may also have beneficial roles in some economic and/or environmental fields, such as wastewater treatment [12] and toxics bioremediation, including fuel and radioactive elements [13, 14]. It has been broadly recognized that biofilm is the preferred and oldest complex growth mode of microorganisms in nature. Indeed, it procures them many advantages. The elevated population density forming a biofilm can increase biological processes that single cells cannot perform. Moreover, the biofilm lifestyle can offer increased protection against environmental stresses (e.g., antimicrobial tolerance and environmental fluctuations such as temperature, pH, and nutrient availability) and expand bacterial survival strategy against host defense responses (phagocytes, etc.) or predators (amoebas, etc.). More, biofilms facilitate horizontal gene transfer [15]. Following the pioneer works of Costerton’s team in the 1970s and 1980s, many investigations have explored the various properties of biofilms in order to better understand

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the behavior of these bacterial communities. Thus, it has been shown that the bacterial phenotype in the biofilm lifestyle is dramatically different across energy conservation, amino acid metabolism, signal transduction, morphogenesis, and many other metabolic pathways compared with the planktonic form. Twenty years after Costerton et al.’s work, Brozel’s team pioneered biofilm proteomics, investigating sessile Bacillus cereus and Pseudomonas aeruginosa proteomes [16, 17]. The proteomics research field consists of large-scale protein study, focusing mainly but not exclusively on expression levels, posttranslational modifications, function, localization, stability, and accumulation, that complements genome sequencing, which alone is of limited value in obtaining a complete understanding of gene function. Proteins being the final products of gene expression, proteomics has become an indispensable additional tool for the global analysis of cellular physiology, in particular for microbial genomes of limited size (less than 10 Mbp for bacteria). Thus, proteomics studies have successfully overcome many dogmas, such as the absence of N-glycosylation in Escherichia coli [18], the absence of enzymes involved in gluconeogenesis in Sulfolobus solfactaricus [19], or the lack of fermentative pathways in Anaeromyxo‑ bacter dehalogenans [20]. The proteome pattern, which is a snapshot of the physiological state, provides invaluable complementary information compared with genomics by characterizing gene product abundances and structures. This need is particularly emphasized by the existence of posttranslational modifications (such as phosphorylation, glycosylation, and ubiquitination), which are often key cellular processes, but lay beyond our reach in genomic investigations. In the present paper, we propose a review of important contributions of proteomics to the knowledge of physiology of sessile bacteria over the last decade.

Biofilm and proteomics Proteomics tools In order to address the proteins specifically recruited within biofilm organism, proteomics investigations on biofilm bacteria emerged rapidly at the beginning of the 2000s. Most of these investigations confirmed older data obtained on artificially immobilized cells (see review [21]), which highlighted changes in metabolic activities and cell resistance in immobilized cells as compared to suspended cultures. In particular, this specific behavior of biofilm organisms has been shown by multivariate analyses, which demonstrated that the biofilm growth mode leads to a specific bacterial proteome in sessile bacteria as compared to their planktonic counterparts.

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Electrophoresis-based proteomic techniques were initially restricted by limitations in resolution and dynamic range. However, these approaches developed rapidly as complementary to transcriptomic analysis, since they allow the detection of functional cellular entities, namely proteins and their PTMs, which cannot be predicted by mRNA expression analysis. Electrophoresis tools (e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDSPAGE], two-dimensional electrophoresis [2D-E], differential in gel electrophoresis [DIGE]) have constituted the first technologies of choice for biofilm proteome studies over the last decades. More recently, highly sensitive mass spectrometry labeling technologies (e.g., stable isotope labeling [SILAC], isotope-coded affinity tag [ICAT], H18 2 O isotope exchange, and isobaric tags for relative and absolute quantitation [iTRAQ]) and label-free quantification became the reference tools in proteomics [22–25] (see Fig. 2). Common labeling techniques consist of modifying proteins/ peptides after direct incorporation of stable isotopes (such as 13C, 15N, or 18O) in the polypeptide sequence or during enzymatic treatment (see different workflows in Fig. 1). The label-free technique involves the integrated measurement of spectral counts or of chromatographic peak areas for any given peptide in successive liquid chromatography–mass spectrometry (LC–MS) runs. Interestingly, label-free quantitation can estimate protein abundance more accurately than gel-based methods and can discriminate the differential protein abundance over a slightly larger dynamic range compared with labeling techniques [26]. However, each of these technologies has its own limitations. Thus, the SILAC approach may induce quantification errors such as an incomplete incorporation of isotopically enriched amino acids [27]. Besides, the label-free approach requires a highly reproducible chromatography separation, and intensity signals may be subject to variation [26]. In fact, both 2D-E- and MS-based methods can reveal interesting molecular features of biofilm development. Strategies implying 2D-E display the capacity to resolve as a mean around 1000 protein spots per gel. For a middlesized bacterial genome coding for 4000 genes, it represents approximatively 15–25 % of the potential proteome. One major added value of 2D-E is to visualize directly on gels the major actors of metabolic pathways, or more importantly, as stated above, the presence of posttranslational modifications. However, in these 2D-E reports, most of the proteome (in particular the proteome of low-abundant proteins) is lacking. Fortunately, more recently developed MS approaches have filled at least partly this gap. These strategies are based on a proteolytic cleavage of the whole protein sample, generating around 100,000 distinct peptides for the same genome (4000 genes). Nowadays, high-resolution MS (HRMS) allows to obtain around 25,000 fragmentation spectra (potential peptide sequence) per hour of

Med Microbiol Immunol Fig. 1 Workflows for biofilm proteomics analysis. Adapted from [204] and published with the authorization of the editor

chromatography gradient, yielding (with longer columns: 50 cm and gradients: 4 h) to a far more exhaustive vision of the biofilm proteome (>80 %). These amazing possibilities enable today to reveal proteins that were never experimentally observed before. However, for the moment, these new approaches have rarely been used for biofilm investigations. As recent examples, SILAC and label-free strategies were employed to compare the proteome of Neisseria gonorrhea, P. aer‑ uginosa, and Staphylococcus epidermidis cells grown in the biofilm and planktonic modes [28–30]. Very recently, an iTRAQ strategy was designed to study the protein profiles of biofilm formation of Streptococcus suis inhibited by erythromycin sub-minimal inhibitory concentration [31]. The biofilm model To compare data obtained in different biofilm studies, it is of major importance to establish a standard laboratory model. Unfortunately, this has not yet been done, and consequently, the literature highlights the diversity of the models used (Table 1). Thus, some research groups considered that traits of colony development could be very similar to those encountered within bacterial biofilms [32]. In a paper

published in 2006, Vilain et al. [33] showed that biofilms are not composed merely of a mixture of planktonic cells at different growth phases; instead, they are physiologically distinct from planktonic cells in exponential, transient and stationary phases. Another experimental question concerns the choice of the control planktonic culture. The majority of the authors chose stationary-phase planktonic cells [34– 39]. Conversely, Mikkelsen et al. [40] reported that the protein profiles of biofilms appeared closely related to those of exponentially growing planktonic organisms. Biofilms and pathogenic bacterial strains It was shown many years ago that biofilm cells are far more resistant to antimicrobials than planktonic counterparts. This observation induced a great interest of scientists for pathogenic bacteria grown in biofilms, whatever they were Gram-negative or Gram-positive organisms. P. aer‑ uginosa is probably the Gram-negative bacterium which was the most studied in biofilms works (see Fig. 2) and so in proteomic investigations [17, 28, 37, 40–54]. Though often proteomically investigated as the canonical bacterial model, pathogenic E. coli strains were also studied [35, 38, 55, 56]. Due to their high involvement in prosthetic

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Table 1 Biofilm models used for proteomics investigations Strain

Planktonic phase

Aggregatibacter actinomy‑ cetemcomitans

Biofilm growth Support and temperature

Analytical tools

References

42 h

24-well cell culture plates, SDS-PAGE, LC MS/MS 37 °C

[106]

Acidithiobacillus. ferrooxidans

24 h

24 h

Pyrite, 28 °C

Shotgun

[165]

Bacillus cereus

2, 18 h Expo, transient, stat

2, 18 h 24 h

Glass wool, 37 °C Glass wool, 30 °C

2D-E, microscopy 2D-E, microscopy

[137] [33]

Bordetella pertussis

Expo, stat

Expo, stat

Flat polypropylene beads, 37 °C

2D-E, MALDI-TOF, FTIR [85]

Bacillus subtilis

18 h

18 h

Glass wool, 37 °C

2D-E, microscopy

[16]

Campylobacter jejuni

Stat (24 h)

24 h

Stainless steel, nitrocellulose, and glass fibers, 37 °C

2D-E, Q-TOF

[39]

20 h

72 h

24-well polystyrene plates, iTRAQ, LC MS/MS 37 °C

Expo, stat

12 h 36 h

Agar-entrapped, 37 °C

N-terminal microsequenc- [205] ing

7 days

7 days

Glass fiber, 20 °C

2D-E, MALDI-TOF, microscopy

16 h

16 h

Synthetic hydrogels, 37 °C 2D-E, LC MS/MS

[38]

Nitrosomonas europaea

Planktonic immobile (3 weeks)

1, 2 weeks

Interface air–liquid in the dark, 28 °C

2D-E, MALDI-TOF

[206]

P. aeruginosa

Stat 24 h

18, 48 h 24 h

Agar-entrapped, 37 °C Polystyrene dishes, anaerobic/aerobic biofilm, 37 °C

2D-E, MALDI-TOF 2D-E, MALDI-TOF

[43] [36]

ICAT

[37]

E. coli

[99]

[35]

Stat

Stat

CF airways

P. putida

1, 4, 6 h

4, 6, 12, 24 h

Silicone/chemostats, room 2D-E, Edman degradation temperature

[88]

Staphylococcus aureus

8, 48 h

8, 48 h

NADIR dialysis membranes, 37 °C

2D-E

[57]

18 h Streptococcus pneumonia

48 h

Heart valve leaflets, 37 °C

2D-E, Q-TRAP

[207]

3, 6, 9 days

Continuous-culture glass, 37 °C

2D-E, MALDI-TOF, microscopy

[98]

X. axonopodis

Early stat

7 days

4-well PVC plates, 28 °C

2D-DIGE, qRT-PCR

[208]

Yersinia ruckeri

48 h

48 h

Agar, 25 °C

2D-E, LC MS/MS

[209]

Expo exponential growth phase, Stat stationary growth phase, h hour

material infections and non-healing wounds, biofilms of Gram-positive bacteria (e.g., Staphylococcus aureus, coagulase-negative Staphylococci, S. epidermidis, and Strep‑ tococcus mutans) were also investigated at the proteomic level [57–70]. Although they are involved in numerous biofilm pathologies, few proteomics studies have focused on the proteome of sessile fungi [71–73]. Characterization of the biofilm proteomes of Candida albicans, Candida glabrata, and Aspergillus fumigatus pointed out an important homology with the behavior of biofilm bacteria, such as enhanced regulation of surface-associated proteins, accumulation of reactive oxygen species, up-regulation of oxidative stress

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response, and increased resistance to commonly used antifungal agents [74–76]. Looking after subproteomes: surfaceome and wall cells proteomes Prokaryotes surfaceomes and cell wall subproteome are very rich in variety and structure. Bacterium surfaceexposed proteome characterization has shed light on the complexity of biological processes taking place at the interface between bacteria and their external milieu. These environment-accessible proteins are not only involved in food intake, fuel, and waste release, but also are also involved


(a) 1800 1600 1400 1200 1000 800 600 400 200 0

(b) 450 400 350

cytosolic one [85]. Obviously, changes in cell size and in the rigidity of the bacterial cell wall were observed by scanning electron microscopy [86]. Other approaches such as genomic, transcriptomic, microscopy, and biochemical tools applied to the study of motility of B. subtilis, P. aeruginosa, V. cholera, and E. coli have demonstrated the same phenomenon [87]. Unfortunately, compared with microscopic and genomic approaches, the subproteomes investigations have been relatively rarely applied for biofilm bacteria because biofilms are often associated with low cell amounts, representing a challenge for further subproteomic studies [51, 77, 88–92] (see Table 2). However, new instrumentation, which allows faster scanning speeds and higher mass accuracy, permits more peptide identifications and so higher proteome coverage, which facilitates the analysis of membrane proteins (see review [93]).

300 250

Biofilm development and proteomics

200 150 100 50 0

Fig.  2 a Bibliometric view of articles developing research on bacterial biofilms (blue) or bacterial biofilm proteins (orange). Data were obtained from a PubMed search (April 2014) with the keywords “bacterial biofilms” or “bacterial biofilm proteins.” b Number of publications including the following terms in the abstract: P. aeruginosa and biofilm associated with transcriptome/proteome/antibiotic/antibiotic resistance/CF/iron/matrix/quorum sensing/antimicrobial/interspecies. Data were also obtained from a PubMed search (January 2014) (color figure online)

in cell mobility, facilitating physical interactions between bacteria and living or inert surfaces, and acting as sensors for extracellular signals and communication. Proteomic studies of subproteomes remain a real challenge due to the hydrophobic nature of integral membrane proteins and their general low abundance levels, imposing the use of many techniques such as compartmentalization, enrichment, fractionation, labeling, biotinylation, shaving of intact cells, and affinity purification [77–84]. The formation of biofilm involves many successive stages that require the regulation of many surface-exposed proteins. Many studies have suggested that the bacterial outer membrane proteome as well as the surfaceome are more affected by the biofilm growth mode than the

Processes of bacterial attachment to living or abiotic surfaces have been investigated at both the molecular and physicochemical levels. Physicochemical mechanisms involved in bacterial adhesion were summarized in a review by Dunne [94]. It is now accepted that the biofilm architecture is heterogeneous and may be divided into intracellular and extracellular domains, although for particular features, a gradient may be present. The biofilm imaging at the micro-, nano-, and mesoscale using laser-based techniques was broadly discussed (see review by Neu and Lawrenc [95]). While it is generally accepted that biofilm development represents a well-organized series of sequential events, the evolution of the bacterial proteome during biofilm development has been moderately characterized (Fig. 3). In 2002, Sauer et al. [96] partitioned the development of P. aerugi‑ nosa biofilm into four stages (i.e., reversible attachment, irreversible attachment, maturation, and detachment) and analyzed each stage by 2D-E. The average difference in proteomes between each developmental stage was around 35 % of detectable proteins, demonstrating that bacteria undergo a global change/remodeling in gene expression following initial attachment to a surface. Using a multivariate statistical approach (principal component analysis, PCA), Vilain et al. confirmed in 2004 that the proteome of attached P. aeruginosa cells differs from that of their planktonic counterparts [97]. This statement was then confirmed by Allegrucci et al. who reported that only 54 % of all the proteins were produced constantly by S. pneumonia cells during biofilm growth [98]. In their paper published in 2004, Vilain et al. showed moreover that the bacterial biofilm phenotype was dependent on the nature of the substratum [97].

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Table 2 Examples of altered proteins in biofilm proteome associated with the surfaceome, membranome, or secretome Strain

Membranome

Surfaceome

Secretome/matrix

References

Gram-negative bacteria Fimbriae

A. actinomycetemcomitans A. baumannii

NlpE, CsuD DcaP-like

C. jejuni

Basal body FlgG, FlgG2, CheA

E. coli

OmpA, D-ribose- and, D-galactose-binding proteins, YbeJ

P. aeruginosa

[210] [144, 211]

Flagellin: FlaA, FlaB, filament cap FliD, Peb1, FlaC Curli, fimbriae 1 Cellulose

[39]

OprF, TonB, lipoprotein OmlA, OprG

PilQ, FliC (decrease)

Alginate, Psl, Pel, EstA, OMVs

[36, 44, 215–217]

P. gingivalis

Outer membrane lipoprotein, HmuY, IhtB, FrdAB

RgpA, HagA, CPG70, PG99

PorSS, LptO

[90]

Pseudoalteromonas sp.

TonB, OmpW, OmpA

Type IV pilus (PilF)

P. putida

NlpD, PotF2 (ABC transport)

S. oneidensis

TolC-like, OmpA

Flagellin, agglutination protein AggA

[141]

T. forsythia

Lipoprotein (TF2843), OmpH

S-layer TfsA and TfsB

[218]

[35, 38, 212–214]

[128] [88]

OMVs

[219]

P. fluorescens

ABC transporter: LapB, LapC, LapA (900 kDa) and LapE

LapA (900 kDa)

[100]

X. axonopodis

Porins FadL, Ton-B dependent Non-fimbrial adhesin: YapH receptor

UDP-glucose dehydrogenase

[208]

Secretome (179 proteins), OMVs

[106]

V. fischeri

A. actinomycetemcomitans N. europaea

OmpR, flagellar basal body rod protein, CheZ, CheW, three porins

FliH, FliG, FliF, FliD, FliM

[206]

Yersinia ruckeri

TolC, OmpA, OmpW, OmpX, TolB, ArtI, DppA, OppA, GlnH, MalE

FlgE, FliC

[209]

N. gonorrhoaeae

OpaD, Omp3, MOMP, FetA, TonB

PilG, PilQ

[29]

SarA, antigen A: IsaA

Fibrinogen-binding proteins: SdrC, SdrD, lipoprotein, β-lactamase, SodB, AhpC, Tuf, Tsf, GlyA, PfkA

Gram-positive bacteria S. aureus

140-kDa antigen, AtlE autolysin, Bap

S. epidermidis B. subtilis

Oligopeptide permease, Vpr

S. pneumonia

[57, 220]

[221]

Flagellin

TasA

[32, 222]

MsrA, enolase, DnaK

UDP-glucose dehydrogenase

[98]

Sec translocase proteins

[223]

L. monocytogenes

Early stages and irreversible attachment Proteome alterations occur rapidly during the initial phases of biofilm formation, and changes may be visible as early as 4 h after initial attachment [88]. Membrane proteins seem to play a major role in bacterial attachment, and in early biofilm development, in particular proteins involved in the interaction with the host [87]. Thus, in C. jejuni, the

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Proteases

first step in adherence to the host cell involves two major cell-binding proteins, i.e., Peb1A and Peb4 [99]. In S. aureus, SdrC and SdrD fibrinogen-binding proteins were also proven to be specifically accumulated in early biofilm growth phases [57]. Proteomic investigations on P. putida, performed 4 and 6 h after initial attachment, additionally showed that a large number of proteins involved in transport, amino acid


Fig. 3 Overview of proteomic contribution to the comprehension of the biofilm phenotype

metabolism, polysaccharides/lipopolysaccharide biosynthesis, motility, DNA replication, antibiotic resistance, and virulence were differentially accumulated [88]. These data suggest that the cell wall is probably the compartment the most altered during these early adhesion steps (see Fig. 3), the quorum sensing (QS) being not involved. Few proteomics studies focused on the irreversible attachment step, which is characterized by the production of extracellular polymeric substances (EPS), i.e., proteins, lipids, lipopolysaccharides (LPS), extracellular DNA, and polysaccharides. In Pseudomonas fluorescens, the transition toward irreversible attachment is mediated by LapA, a large secreted protein that is associated with the bacterium surface [100]. In P. aeruginosa, this step requires the BfiSR two-component system, as well as small regulatory RNA, specifically CafA [101]. Biofilm maturation During this stage, the biofilm acquires a three-dimensional architecture which differs significantly among bacterial strains, as well as between serotypes of the same strain [98].

Singularity of the mature biofilm phenotype Within mature biofilms, bacteria exhibit a specific proteome that is statistically different from those of both exponential- and stationary-phase planktonic organisms [33]. Multivariate methods have been used to interpret the variations in spot densities observed on the proteomic map from P. aeruginosa cell cultures as suspensions or biofilms on different supports. This PCA extracted three significant components, explaining 78.4 % of the variability in the data. A first component discriminated free cell cultures and biofilm ones, arguing for the specificity of the biofilm phenotype. A second one was essentially related to free cell cultures, whereas the third component was assigned to the substratum nature [97]. These observations accord well with other investigations stressing the role of the substratum nature on the resistance of sessile bacteria to antimicrobials [102–104]. To test whether the biofilm population was merely composed of a mixture of planktonic cells at different growth stages, B. cereus biofilm proteome was compared to those of exponential-, transient-, or stationary-phase planktonic

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populations [33]. This analysis demonstrated that the biofilm proteome was unique and not only a mixture of proteins present in exponential- and stationary-phase bacteria. Matrix proteins Up to now, few proteomics investigations have been devoted to matrix exoproteome. The extracellular matrix fraction of Haemophilus influenzae biofilm was subjected to proteomic analysis [105]. The study allowed the identification of 265 proteins such as GroEL, KasA/FabB, OmpA, UspA, and peroxiredoxin. Many of these proteins were hypothetical or uncharacterized. A proteomic study performed on the secretome of sessile Aggregatibacter actino‑ mycetemcomitans cells pointed out the accumulation of virulence factors (e.g., DegQ, fHbp, LppC, Mip, NlpB, Pcp, PotD, TolB, and TolC), pointing out the clinical interest of such study [106]. A proteomic analysis performed on P. aeruginosa biofilm matrix showed that approximately 30 % of the identified matrix proteins are OMPs, which are also present in outer membrane vesicles (OMVs) [51]. The role of OMVs in the biofilm matrix has been discussed [107, 108], but remains under debate [109–111]. It looks like the majority of matrix proteins could be encapsulated in OMVs, even though cell lysis cannot be completely excluded. For instance, these vesicles can ensure the transport of periplasmic proteins from one bacterium to another to share antibiotic resistance [112]. A proteomic investigation showed that planktonic P. aeruginosa cells produced OMVs that play a role in pathogenesis [113]. Conversely, virulence factors seem to be at low level in biofilm OMVs, whereas proteins involved in iron acquisition are accumulated [51]. In Helicobacter pylori, OMVs might be involved in the strain biofilm-forming ability, particularly through the up-regulation of a unique 22-kDa protein [107, 114]. In another species such as V. cholera, DegP, a OMVs protein, is required for the secretion of biofilm matrix components and substrates of type II secretion system [111]. Detachment phase In the final step of the biofilm lifecycle, some bacteria leave the consortium to seek for colonization of new surfaces. This dispersion could be either an active process leading to the production of differentiated highly motile cells known as dispersal cells, or a passive dispersal, resulting from sloughing of cells and erosion from the biofilm (see review [115]). Active dispersal from biofilms often proceeds through localized death and cell lysis [116]. Additionally, roles for CsrA (a carbon storage regulator) and flagella in the dispersal of the biofilm have been suggested [117]. An

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increase in flagellin (FliC) expression and the decrease in that of pilA were confirmed by a genomic approach [42]. This phenomenon can be promoted by sudden increases in substrate availability, such as succinate, glutamate, or glucose, which led to approximately 80 % reductions in surface-associated biomass [42]. Besides, Huynh et al. [118] characterized the dispersal of biofilm by quantifying changes in the proteome of P. aeruginosa biofilm compared with planktonic cells during glucose starvation using an iTRAQ approach. The dispersion started within 5 min of glucose starvation was maximal after 2 h, up to 60 % of the original biomass being dispersed after 24 h of starvation. This proteomic analysis revealed that more than 100 proteins (belonging to various functional classes including carbon and energy metabolism, stress adaptation, and motility) were differentially expressed when the bacteria prepared their metabolism for future planktonic survival. An outstanding result was the first demonstration that starvation-induced dispersal in P. aeruginosa operates through the intracellular second messenger cAMP and that dispersal is an active process, involving many receptors, effectors, and regulatory proteins [118]. More, in Mar‑ inobacter hydrocarbonoclasticus, the comparison of the proteome of dispersed cells with that of biofilm or planktonic cells indicated that dispersed cells has lost most of the biofilm phenotype and expressed a specific proteome [119, 120]. Lately, the emergent concept that QS activates the biofilm dispersion process was reviewed [121]. Beside, recent evidence suggests that regulatory networks governing the decision of bacteria whether to attach and form biofilms or remain as planktonic cells are further subject to regulation by small noncoding RNAs (see review [122]).

Living in a bacterial community Metaproteomics In nature, biofilms are rarely formed by a single bacterial species, but rather by multiple species that interact/compete with each other. This field of research is called metaproteomics, or environmental proteomics, and allows key functions to be attributed to specific bacterial members, and to reveal cooperation events. A first metaproteomic study was performed within a natural biofilm community growing in acid mine drainage at Iron Mountain near California [123]. Profiling with shotgun proteomics identified more than 2000 proteins from the five most abundant species. Half of these proteins were derived from the prevailing organism in this environment, Leptospirillum group II. Nowadays, the metaproteomic study of many ecosystems is emerging. For example, human intestinal microbiota, crop rhizospheric soil, sludge,


marine biofilms, and biofilms associated with acute apical abscesses and asymptomatic apical periodontitis lesions have been recently investigated [124–129]. Multispecies communities Communication mediated by secreted diffusible molecules is widespread among bacteria and regulates several important physiological and virulence-related properties, including biofilm formation. Indeed, it was observed synergetic effects of four strains isolated from the surface of the marine alga promote biofilm biomass by 167 % and resistance to antimicrobial agents [130] compared with axenic biofilms. In addition, in a recent proteomic study, which Porphyromonas gingivalis, Treponema denticola, and T. forsythia were cultured in a polymicrobial biofilm, ZainalAbidin et al. [130, 131] identified many proteins involved in bacterial interactions. Their data also suggested a change in the strategy of iron acquisition by P. gingivalis, with an increase in abundance of HusA and HusB, while HmuY and other iron/heme transport systems decreased. Interestingly, this work suggested an intimate association between P. gingivalis and T. denticola that may play a role in pathogenesis [131]. Signaling and communication In the last decades, the availability of highly sensitive analytical strategies has boosted the characterization of molecular events occurring within these microbial consortia and signaling network regulators within biofilms. Quorum sensing Bacteria commonly communicate with each other by a cellto-cell signaling mechanism, known as quorum sensing (QS), which is a major actor during biofilm development [121, 132]. This bacterial intercellular signaling involves the production, detection, and response to extracellular signaling molecules called autoinducers (AIs). AIs accumulate in the extracellular environment as the bacterial population density increases, and bacteria integrate this signal to track changes in their population density and collectively alter gene expression [133]. Gram-positive and Gramnegative bacteria use different types of QS systems: Grampositive bacteria use peptides, called autoinducing peptides (AIPs), whereas Gram-negative bacteria communicate using AHLs or other molecules whose production depends on S-adenosyl methionine (SAM) as substrate [134]. For instance, P. aeruginosa harbors three QS systems, namely two LuxI/LuxR-type QS circuits and a third non-LuxI/ LuxR-type system called the Pseudomonas quinolone signal (PQS) system [134]. Some examples are LasI/LasR and

RhlI/RhlR in P. aeruginosa [135]. The involvement of QS systems in biofilm formation has been well documented [136]. In an interesting design, Arevalo-Ferro and coworkers compared the protein patterns of the intracellular, extracellular, and surface protein fractions of the P. aeruginosa PAO1 parent strain with those of an isogenic lasI rhlI double mutant. This works highlighted numerous proteins under the control of QS that were associated with iron utilization [41]. In addition, the comparative proteome analysis of a wild-type P. putida strain and a QS-deficient mutant grown either in planktonic cultures or in biofilms showed that more than half of identified surface proteins were regulated by the ppu QS system [45]. By using a proteomic approach, it was also demonstrated that biofilm P. aerugi‑ nosa cells might modify the physiology of planktonic surrounding cells previously called SIP (surface-influenced planktonic) [137]. Indeed, planktonic cells could readily detect the presence of a biofilm in their close environment and modify their gene expression accordingly [48]. However, the mechanisms, which are involved, remain to be determined. For Gram-positive bacteria, S. aureus biofilms produced greater amounts of a non-ribosomal peptide (phevalin) compared with their planktonic counterparts. Surprisingly, the effect of phevalin alone on the S. aureus extracellular proteome and metabolome was low [138]. The commensal S. epidermis species secretes an enzyme called Esp (serine protease) that is able to disassemble preformed S. aureus biofilm and inhibit its colonization capacity [139]. A proteomics analysis showed that Esp inhibits S. aureus cells colonization and biofilm formation by degrading crucial specific proteins for biofilm matrix, such as extracellular adherence protein, fibronectin-binding protein A, protein A [140]. Iron Early proteomic studies have suggested that iron availability is important for biofilm development [41, 68, 123, 141]. Under iron-limiting circumstances, bacteria can import heme or heme-containing compounds and use them as an iron source. More, under iron starvation conditions, aerobic bacteria synthesize and secrete siderophores into the extracellular environment. In P. aeruginosa, these iron regulatory systems are controlled by the QS [41]. The ferric siderophore complexes are transported into the cells by iron OMP systems, TonB, and two accessory proteins, ExbB and ExbD [142]. In vitro metaproteomic analysis of P. aeruginosa and Morganella morganii isolated from a catheter-associated biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of

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corresponding receptors [143]. Moreover, biofilm proteome in many bacteria such as A. baumannii and Streptococcus mutans has been shown to accumulate protein involved in Fe transport or [Fe-S] clusters formation [68, 144]. A more focused investigation of the membrane subproteome in A. baumannii demonstrated that the development of a biofilm at the air–liquid interface was promoted by ferric ions across the overexpression of four siderophore iron uptake systems [145]. Cyclic diguanosine‑5′‑monophosphate (c‑di‑GMP) C-di-GMP is widely described as a universal molecule for biofilm initiation and formation. In fact, a high intracellular level of c-di-GMP induces the biofilm lifestyle, whereas a low intracellular level stimulates biofilm dispersal and promotes the planktonic lifestyle [146–148]. The intracellular level of c-di-GMP is controlled through opposing diguanylate cyclase and phosphodiesterase activities; these enzymes contain GGDEF and EAL domain proteins, respectively. Proteomics played a crucial role in deciphering c-di-GMP regulatory effects and interacting proteins [53, 149]. In particular, it was reported that low intracellular c-di-GMP level in cells dispersed from biofilms induced the expression of proteins required for virulence and the development of antimicrobial peptide resistance in P. aer‑ uginosa. In accordance with this observation, P. aerugi‑ nosa cells with low c-di-GMP levels were found to be more resistant to colistin than cells with high c-di-GMP levels [150]. Signal transduction network Transcriptional regulators The regulation of bacterium metabolism requires proteins involved in signal transduction, which allows the sensing of environmental fluctuations (e.g., temperature, pH, nutrient availability). In E. coli, the Cpx transduction system is involved in surface sensing and adhesion and in the modulation of the expression of curli [151]. In P. tunicata, the ToxRlike regulator WmpR was shown to regulate the expression of bioactive compounds, type IV pili, and biofilm formation. Transcriptomic and proteomic analyses in the ΔwmpR mutant suggested that WmpR controls the expression of genes encoding proteins involved in iron uptake, amino acid metabolism, and ubiquinone biosynthesis, in addition to a number of proteins with unknown function [152]. Sigma factors Alternative sigma factors play critical roles in the ability of bacteria to adapt to diverse environmental conditions and to form biofilms [153–155]. In 2003, Schembri et al. reported that 46 % of E. coli genes, which were

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differentially expressed during biofilm growth, are under the control of the δS subunit of RpoS. Moreover, deletion of rpoS made E. coli nearly inept of establishing sessile communities [156]. In order to investigate the role of rpoS, Collet et al. [55] compared the proteomes of an E. coli rpoS mutant with those of the wild-type strain. Scanning electron microscopy (SEM) showed that the rpoS deletion altered biofilm architecture and adherent cell density. This investigation demonstrated that rpoS plays a role in the special nature of the gene expression of biofilm cells but to a lesser extent than in stationary-phase planktonic bacteria. Interestingly, some RpoS-regulated proteins were different, depending on the culture condition: planktonic or biofilm [55]. As an intriguing difference, among Gram-negative bacteria, RpoS seems to be less important for the P. aeruginosa biofilm lifecycle [157]. However, proteomics approaches suggest that sigma54 factor (RpoN) seems important for biofilm formation of P. aeruginosa and other bacteria such as Legionella pneu‑ mophila, Enterococcus faecalis, Burkholderia cenocepacia, and Vibrio anguillarum [154, 158–161].

Perspective Proteomics, biofilm, and highly regulated hypothetical proteins: a close relationship Considering that for more than five decades, we thought that the privileged growth mode of bacteria was the planktonic mode, most investigations performed were conducted on free-floating bacteria. Those thousands of investigations constituted the basis for proteins functional annotation. The main consequence is that most biofilm-specific proteins (i.e., proteins only produced by sessile organisms) still have unknown or hypothetical functions. Therefore, in a recent study, the proteomes of Desulfovibrio vulgaris and Vibrio parhaemolyticus cells grown in the sessile and planktonic modes showed that the majority of proteins accumulated by biofilm cells were hypothetical [162, 163]. Even, in species as P. aeruginosa, which has a very wellannotated genome, the characterization of whole-cell biofilm proteome using a high-performance spectrometry tool highlighted the accumulation of multiple highly prominent hypothetical/unknown proteins that could play a significant role in biofilm structure or development [28]. An example of such orphan system involved in the biofilm formation is the biofilm-associated cluster (bac) system, which was shown to be involved in the biofilm formation ability of P. aeruginosa [164]. In 2012, Vera et al. [165] performed a high-throughput proteomic investigation and tried to annotate several unknown proteins involved in the early biofilm formation in A. ferrooxidans. More recently, the number of uncharacterized proteins following proteomic analysis


by MS high-sensitivity technologies has considerably increased. In fact, unfortunately and after three decades of biofilm research, these proteins/genes remain to be associated with their specific role within biofilms [166]. Posttranslational modifications (PTMs) The proteomics of biofilm has developed extensively over the last decade. However, investigations have neglected PTMs-associated proteins, which are most often stacked in the immersed part of the proteomic iceberg. In fact, PTMs remain inaccessible to genomic and transcriptomic analyses because they are neither detectable nor predictable from gene sequences or messenger RNA expression levels. Many studies have suggested that PTMs play a key role in biofilm developmental steps [101, 167–175]. The major bacterial PTMs described to date are phosphorylation, glycosylation, and acetylation [176]. Nevertheless, other types of investigations need to be performed to assess potential modifications more exhaustively. Despite the high importance of phosphorylation in the bacterial physiology, only few phosphoproteomics investigations have been performed on biofilm cells [177, 178]. The two-component signal transduction systems (TCSTSs) are probably the phosphoprotein-involving systems that have been mostly investigated in the biofilm framework. Many of these TCSTSs have been shown to participate in biofilm formation [101, 179–181]. In 2009, Petrova and Sauer [178] investigated the phosphoproteome of P. aerugi‑ nosa at different stages of biofilm development via 2D-E technology associated with immunoblotting and cleavable isotope-coded affinity tag (cICAT) MS to describe a novel committed signal transduction network regulating biofilm’s developmental steps following attachment. Three novel P. aeruginosa TCSTSs were highlighted in regulating the transition to irreversible attachment (BfiRS, stage 1–2), maturation-1 (BfmRS, stage 2–3), and maturation-2 (MifRS, stage 3–4) during biofilm development in response to as yet unknown intra- and/or extracellular signals. Phosphorylation occurring in a sequential manner, this suggests the presence of a TCSTS network during the progression of biofilm development in response to environmental cues. Amazingly, in another report also dealing with P. aerugi‑ nosa, nutrient-induced bacteria dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins of the newly dispersed cells, the inhibition of dephosphorylation reducing the extent of dispersion cell from the biofilm [42]. In glycoproteomics, the key structural issue is protein identification, glycosylation site determination, and glycan profiling. Despite the importance of this PTM in cellular physiology, few glycoproteomics studies have been

devoted to prokaryotes [182–188], and even fewer to biofilm investigations [182]. Several reasons may be advanced to explain this discrepancy: (1) the view that bacteria are unable to synthesize glycoproteins, which existed for a long time [189], (2) the low abundance of glycoforms, and (3) the technical complexity of characterizing glycosylation. Today, we know that glycosylation is often correlated in bacteria with pathogenicity, and N-glycosylation is particularly prevalent in proteins destined for extracellular environments [190, 191]. Notably, glycosylated proteins bear key signaling for host/microorganism interactions and bacterial pathogenicity (S-layers, antigen, pilin, flagellin) [192–194] and are also involved in adhesive functions [167, 195]. It is noteworthy that proteomic demonstration of the role of glycoproteins in biofilm formation was demonstrated in fungi [196, 197]. Biofilm and environmental processes Although frequently associated with disease and biofouling, biofilms are also important for engineering applications such as bioremediation, biomineralization, biocatalysis, and microbial fuel cells (MFC). Bacteria remediate contaminated environments and produce electricity via bioconversion of wastewater. Indeed, organic waste substrates have been well investigated in recent years. Moreover, electrochemically active biofilms in bio-electrochemical systems have received a great deal of attention [198, 199]. Unfortunately, proteomics investigations of these kinds of bacteria and their associated beneficial environmental applications have been much less numerous [200–203].

Conclusion Although biofilms probably constitute the prominent growth mode of bacteria in their natural environment, the planktonic model remains the model of choice for microbiology investigations. This is probably a habit that developed from Pasteur’s works. Consequently, although bacteria have been observed since the seventeenth century, biofilms have been deeply investigated only since the last 15 years. Today, the remarkable progress realized in mass spectrometry and bioinformatics allows this sessile world to be investigated in depth at the protein level. We can consequently advance that proteomics will bring new relevant information about the physiology of biofilm organisms in the very near future. Acknowledgments Grant: program number 33267 FEDER/CNRS. Conflict of interest The authors report no declaration of interest.

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References 1. Nickel JC, Ruseska I, Wright JB, Costerton JW (1985) Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob Agents Chemother 27(4):619–624 2. Hoyle BD, Costerton JW (1991) Bacterial resistance to antibiotics: the role of biofilms. Prog Drug Res 37:91–105 3. Zegans ME, Becker HI, Budzik J, O’Toole G (2002) The role of bacterial biofilms in ocular infections. DNA Cell Biol 21(5– 6):415–420. doi:10.1089/10445490260099700 4. Costerton JW, Montanaro L, Arciola CR (2005) Biofilm in implant infections: its production and regulation. Int J Artif Organs 28(11):1062–1068 5. Zottola EA, Sasahara KC (1994) Microbial biofilms in the food processing industry—Should they be a concern? Int J Food Microbiol 23(2):125–148. doi: 10.1016/0168-1605(94)90047-7 6. Kumar CG, Anand SK (1998) Significance of microbial biofilms in food industry: a review. Int J Food Microbiol 42(1– 2):9–27. doi:10.1016/S0168-1605(98)00060-9 7. Schlegelova J, Karpiskova S (2007) Microbial biofilms in the food industry. Epidemiol Mikrobiol Imunol 56(1):14–19 8. Rodrigues CM, Takita MA, Coletta-Filho HD, Olivato JC, Caserta R, Machado MA, de Souza AA (2008) Copper resistance of biofilm cells of the plant pathogen Xylella fastidiosa. Appl Microbiol Biotechnol 77(5):1145–1157. doi:10.1007/ s00253-007-1232-1 9. Akuzov D, Brummer F, Vladkova T (2013) Some possibilities to reduce the biofilm formation on transparent siloxane coatings. Colloids Surf B Biointerfaces 104:303–310. doi:10.1016/j. colsurfb.2012.09.036 10. Dussart L, Dupont JP, Zimmerlin I, Lacroix M, Saiter JM, Junter GA, Jouenne T (2003) Occurrence of sessile Pseudomonas oryzihabitans from a karstified chalk aquifer. Water Res 37(7):1593–1600. doi:10.1016/ S0043-1354(02)00555-9 11. Abram F, Gunnigle E, O’Flaherty V (2009) Optimisation of protein extraction and 2-DE for metaproteomics of microbial communities from anaerobic wastewater treatment biofilms. Electrophoresis 30(23):4149–4151. doi:10.1002/elps.200900474 12. Martin KJ, Nerenberg R (2012) The membrane biofilm reactor (MBfR) for water and wastewater treatment: principles, applications, and recent developments. Bioresour Technol 122:83– 94. doi:10.1016/j.biortech.2012.02.110 13. Sarro MI, Garcia AM, Moreno DA (2005) Biofilm formation in spent nuclear fuel pools and bioremediation of radioactive water. Int Microbiol 8(3):223–230. doi:10.1007/ s10295-007-0215-7 14. Katuri KP, Enright AM, O’Flaherty V, Leech D (2012) Microbial analysis of anodic biofilm in a microbial fuel cell using slaughterhouse wastewater. Bioelectrochemistry 87:164–171. doi:10.1016/j.bioelechem.2011.12.002 15. Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284(5418):1318–1322 16. Oosthuizen MC, Steyn B, Lindsay D, Brozel VS, von Holy A (2001) Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm. FEMS Microbiol Lett 194(1):47–51. doi:10.1111/j.1574-6968.2001.tb09444.x 17. Steyn B, Oosthuizen MC, MacDonald R, Theron J, Brozel VS (2001) The use of glass wool as an attachment surface for studying phenotypic changes in Pseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis. Proteomics 1(7):871–879. doi:10.1002/1615-9861(200107)1:73.0.CO;2-2

13

Med Microbiol Immunol 18. Pandhal J, Ow SY, Noirel J, Wright PC (2011) Improving N-glycosylation efficiency in Escherichia coli using shotgun proteomics, metabolic network analysis, and selective reaction monitoring. Biotechnol Bioeng 108(4):902–912. doi:10.1002/ bit.23011 19. Barry RC, Young MJ, Stedman KM, Dratz EA (2006) Proteomic mapping of the hyperthermophilic and acidophilic archaeon Sulfolobus solfataricus P2. Electrophoresis 27(14):2970–2983. doi:10.1002/elps.200500851 20. Chao TC, Kalinowski J, Nyalwidhe J, Hansmeier N (2010) Comprehensive proteome profiling of the Fe(III)-reducing myxobacterium Anaeromyxobacter dehalogenans 2CP-C during growth with fumarate and ferric citrate. Proteomics 10(8):1673– 1684. doi:10.1002/pmic.200900687 21. Junter GA, Jouenne T (2004) Immobilized viable microbial cells: from the process to the proteome em leader or the cart before the horse. Biotechnol Adv 22(8):633–658. doi:10.1016/j. biotechadv.2004.06.003 22. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17(10):994–999 23. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1(5):376–386 24. Staes A, Demol H, Van Damme J, Martens L, Vandekerckhove J, Gevaert K (2004) Global differential non-gel proteomics by quantitative and stable labeling of tryptic peptides with oxygen-18. J Proteome Res 3(4):786–791 25. Wiese S, Reidegeld KA, Meyer HE, Warscheid B (2007) Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics 7(3):340–350. doi:10.1002/pmic.200600422 26. Neilson KA, Ali NA, Muralidharan S, Mirzaei M, Mariani M, Assadourian G, Lee A, van Sluyter SC, Haynes PA (2011) Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics 11(4):535–553. doi:10.1002/ pmic.201000553 27. Park SS, Wu WW, Zhou Y, Shen RF, Martin B, Maudsley S (2012) Effective correction of experimental errors in quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC). J Proteomics 75(12):3720–3732. doi:10.1016/j.jprot.2012.04.035 28. Park AJ, Murphy K, Krieger JR, Brewer D, Taylor P, Habash M, Khursigara CM (2014) A temporal examination of the planktonic and biofilm proteome of whole cell Pseudomonas aer‑ uginosa PAO1 using quantitative mass spectrometry. Mol Cell Proteomics. doi:10.1074/mcp.M113.033985 29. Phillips NJ, Steichen CT, Schilling B, Post DM, Niles RK, Bair TB, Falsetta ML, Apicella MA, Gibson BW (2012) Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins. PLoS ONE 7(6):e38303. doi:10.1371/journal.pone.0038303 30. Carvalhais V, Cerca N, Vilanova M, Vitorino R (2015) Proteomic profile of dormancy within Staphylococcus epidermidis biofilms using iTRAQ and label-free strategies. Appl Microbiol Biotechnol. doi:10.1007/s00253-015-6434-3 31. Zhao YL, Zhou YH, Chen JQ, Huang QY, Han Q, Liu B, Cheng GD, Li YH (2015) Quantitative proteomic analysis of sub-MIC erythromycin inhibiting biofilm formation of S. suis in vitro. J Proteomics 116:1–14. doi:10.1016/j.jprot.2014.12.019 32. Morikawa M, Kagihiro S, Haruki M, Takano K, Branda S, Kolter R, Kanaya S (2006) Biofilm formation by a Bacillus sub‑ tilis strain that produces gamma-polyglutamate. Microbiology 152(Pt 9):2801–2807. doi:10.1099/mic.0.29060-0

Med Microbiol Immunol 33. Vilain S, Brozel VS (2006) Multivariate approach to comparing whole-cell proteomes of Bacillus cereus indicates a biofilm-specific proteome. J Proteome Res 5(8):1924–1930. doi:10.1021/ pr050402b 34. Stoodley P, Sauer K, Davies DG, Costerton JW (2002) Biofilms as complex differentiated communities. Annu Rev Microbiol 56:187–209. doi:10.1146/annurev.micro.56.012302.160705 35. Tremoulet F, Duche O, Namane A, Martinie B, Labadie JC (2002) A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode. FEMS Microbiol Lett 215(1):7–14. doi:10.1111/j.1574-6968.2002.tb11363.x 36. Yoon SS, Hennigan RF, Hilliard GM, Ochsner UA, Parvatiyar K, Kamani MC, Allen HL, DeKievit TR, Gardner PR, Schwab U, Rowe JJ, Iglewski BH, McDermott TR, Mason RP, Wozniak DJ, Hancock RE, Parsek MR, Noah TL, Boucher RC, Hassett DJ (2002) Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis. Dev Cell 3(4):593–603. doi:10.1016/S1534-5807(02)00295-2 37. Guina T, Purvine SO, Yi EC, Eng J, Goodlett DR, Aebersold R, Miller SI (2003) Quantitative proteomic analysis indicates increased synthesis of a quinolone by Pseudomonas aeruginosa isolates from cystic fibrosis airways. Proc Natl Acad Sci USA 100(5):2771–2776. doi:10.1073/pnas.0435846100 38. Orme R, Douglas CW, Rimmer S, Webb M (2006) Proteomic analysis of Escherichia coli biofilms reveals the overexpression of the outer membrane protein OmpA. Proteomics 6(15):4269– 4277. doi:10.1002/pmic.200600193 39. Kalmokoff M, Lanthier P, Tremblay TL, Foss M, Lau PC, Sanders G, Austin J, Kelly J, Szymanski CM (2006) Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation. J Bacteriol 188(12):4312–4320. doi:10.1128/JB.01975-05 40. Mikkelsen H, Duck Z, Lilley KS, Welch M (2007) Interrelationships between colonies, biofilms, and planktonic cells of Pseudomonas aeruginosa. J Bacteriol 189(6):2411–2416. doi:10.1128/JB.01687-06 41. Arevalo-Ferro C, Hentzer M, Reil G, Gorg A, Kjelleberg S, Givskov M, Riedel K, Eberl L (2003) Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics. Environ Microbiol 5(12):1350–1369. doi:10.1046/j.1462-2920.2003.00532.x 42. Sauer K, Cullen MC, Rickard AH, Zeef LA, Davies DG, Gilbert P (2004) Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J Bacteriol 186(21):7312–7326. doi:10.1128/JB.186.21.7312-7326.2004 43. Vilain S, Cosette P, Hubert M, Lange C, Junter GA, Jouenne T (2004) Comparative proteomic analysis of planktonic and immobilized Pseudomonas aeruginosa cells: a multivariate statistical approach. Anal Biochem 329(1):120–130. doi:10.1016/j. ab.2004.02.014 44. Seyer D, Cosette P, Siroy A, Dé E, Lenz C, Vaudry H, Coquet L, Jouenne T (2005) Proteomic comparison of outer membrane protein patterns of sessile and planktonic Pseudomonas aeruginosa cells. Biofilms 2(01):27–36. doi:10.1017/ S1479050505001638 45. Arevalo-Ferro C, Reil G, Gorg A, Eberl L, Riedel K (2005) Biofilm formation of Pseudomonas putida IsoF: the role of quorum sensing as assessed by proteomics. Syst Appl Microbiol 28(2):87–114. doi:10.1016/j.syapm.2004.10.005 46. Thompson MR, Chourey K, Froelich JM, Erickson BK, VerBerkmoes NC, Hettich RL (2008) Experimental approach for deep proteome measurements from small-scale microbial biomass samples. Anal Chem 80(24):9517–9525. doi:10.1021/ ac801707s

47. Mikkelsen H, Bond NJ, Skindersoe ME, Givskov M, Lilley KS, Welch M (2009) Biofilms and type III secretion are not mutually exclusive in Pseudomonas aeruginosa. Microbiology 155(Pt 3):687–698. doi:10.1099/mic.0.025551-0 48. Nigaud Y, Cosette P, Collet A, Song PC, Vaudry D, Vaudry H, Junter GA, Jouenne T (2010) Biofilm-induced modifications in the proteome of Pseudomonas aeruginosa planktonic cells. Biochim Biophys Acta 1804(4):957–966. doi:10.1016/j. bbapap.2010.01.008 49. Varela C, Mauriaca C, Paradela A, Albar JP, Jerez CA, Chavez FP (2010) New structural and functional defects in polyphosphate deficient bacteria: a cellular and proteomic study. BMC Microbiol 10:7. doi:10.1186/1471-2180-10-7 50. Benamara H, Rihouey C, Jouenne T, Alexandre S (2011) Impact of the biofilm mode of growth on the inner membrane phospholipid composition and lipid domains in Pseudomonas aer‑ uginosa. Biochim Biophys Acta 1808(1):98–105. doi:10.1016/j. bbamem.2010.09.004 51. Toyofuku M, Roschitzki B, Riedel K, Eberl L (2012) Identification of proteins associated with the Pseudomonas aeruginosa biofilm extracellular matrix. J Proteome Res 11(10):4906–4915. doi:10.1021/pr300395j 52. Petrova OE, Schurr JR, Schurr MJ, Sauer K (2012) Microcolony formation by the opportunistic pathogen Pseudomonas aeruginosa requires pyruvate and pyruvate fermentation. Mol Microbiol 86(4):819–835. doi:10.1111/mmi.12018 53. Nesper J, Reinders A, Glatter T, Schmidt A, Jenal U (2012) A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins. J Proteomics 75(15):4874– 4878. doi:10.1016/j.jprot.2012.05.033 54. Chellappa ST, Maredia R, Phipps K, Haskins WE, Weitao T (2013) Motility of Pseudomonas aeruginosa contributes to SOS-inducible biofilm formation. Res Microbiol 164(10):1019– 1027. doi:10.1016/j.resmic.2013.10.001 55. Collet A, Cosette P, Beloin C, Ghigo JM, Rihouey C, Lerouge P, Junter GA, Jouenne T (2008) Impact of rpoS deletion on the proteome of Escherichia coli grown planktonically and as biofilm. J Proteome Res 7(11):4659–4669. doi:10.1021/pr8001723 56. Mukherjee J, Ow SY, Noirel J, Biggs CA (2011) Quantitative protein expression and cell surface characteristics of Escheri‑ chia coli MG1655 biofilms. Proteomics 11(3):339–351. doi:10.1002/pmic.201000386 57. Resch A, Leicht S, Saric M, Pasztor L, Jakob A, Gotz F, Nordheim A (2006) Comparative proteome analysis of Staphy‑ lococcus aureus biofilm and planktonic cells and correlation with transcriptome profiling. Proteomics 6(6):1867–1877. doi:10.1002/pmic.200500531 58. Veenstra GJ, Cremers FF, van Dijk H, Fleer A (1996) Ultrastructural organization and regulation of a biomaterial adhesin of Staphylococcus epidermidis. J Bacteriol 178(2):537–541 59. Wagner C, Kondella K, Bernschneider T, Heppert V, Wentzensen A, Hansch GM (2003) Post-traumatic osteomyelitis: analysis of inflammatory cells recruited into the site of infection. Shock 20(6):503–510. doi:10.1097/01.shk.0000093542.78705. e3 60. Xu L, Li H, Vuong C, Vadyvaloo V, Wang J, Yao Y, Otto M, Gao Q (2006) Role of the luxS quorum-sensing system in biofilm formation and virulence of Staphylococcus epidermidis. Infect Immun 74(1):488–496. doi:10.1128/IAI.74.1.488-496.2006 61. Batzilla CF, Rachid S, Engelmann S, Hecker M, Hacker J, Ziebuhr W (2006) Impact of the accessory gene regulatory system (Agr) on extracellular proteins, codY expression and amino acid metabolism in Staphylococcus epidermidis. Proteomics 6(12):3602–3613. doi:10.1002/pmic.200500732

13

62. Kong KF, Vuong C, Otto M (2006) Staphylococcus quorum sensing in biofilm formation and infection. Int J Med Microbiol 296(2–3):133–139. doi:10.1016/j.ijmm.2006.01.042 63. Qin Z, Yang X, Yang L, Jiang J, Ou Y, Molin S, Qu D (2007) Formation and properties of in vitro biofilms of ica-negative Staphylococcus epidermidis clinical isolates. J Med Microbiol 56(Pt 1):83–93. doi:10.1099/jmm.0.46799-0 64. Brooks JL, Jefferson KK (2012) Staphylococcal biofilms: quest for the magic bullet. Adv Appl Microbiol 81:63–87. doi:10.1016/B978-0-12-394382-8.00002-2 65. Arciola CR, Campoccia D, Speziale P, Montanaro L, Costerton JW (2012) Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials. Biomaterials 33(26):5967–5982. doi:10.1016/j.biomaterials.2012.05.031 66. Otto M (2014) Staphylococcus epidermidis pathogenesis. Methods Mol Biol 1106:17–31. doi:10.1007/978-1-62703-736-5_2 67. McNeill K, Hamilton IR (2004) Effect of acid stress on the physiology of biofilm cells of Streptococcus mutans. Microbiology 150(Pt 3):735–742 68. Rathsam C, Eaton RE, Simpson CL, Browne GV, Valova VA, Harty DW, Jacques NA (2005) Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms. J Proteome Res 4(6):2161–2173. doi:10.1021/ pr0502471 69. Khan AU, Islam B, Khan SN, Akram M (2011) A proteomic approach for exploring biofilm in Streptococcus mutans. Bioinformation 5(10):440–445 70. Li J, Wang W, Wang Y, Zeng AP (2013) Two-dimensional gelbased proteomic of the caries causative bacterium Streptococ‑ cus mutans UA159 and insight into the inhibitory effect of carolacton. Proteomics 13(23–24):3470–3477. doi:10.1002/ pmic.201300077 71. Mukherjee PK, Chandra J (2004) Candida biofilm resistance. Drug Resist Updates 7(4–5):301–309. doi:10.1016/j. drup.2004.09.002 72. Chandra J, Zhou G, Ghannoum MA (2005) Fungal biofilms and antimycotics. Curr Drug Targets 6(8):887–894 73. Muszkieta L, Beauvais A, Pahtz V, Gibbons JG, Anton Leberre V, Beau R, Shibuya K, Rokas A, Francois JM, Kniemeyer O, Brakhage AA, Latge JP (2013) Investigation of Aspergillus fumigatus biofilm formation by various “omics” approaches. Front Microbiol 4:13. doi:10.3389/fmicb.2013.00013 74. Pierce CG, Thomas DP, Lopez-Ribot JL (2009) Effect of tunicamycin on Candida albicans biofilm formation and maintenance. J Antimicrob Chemother 63(3):473–479. doi:10.1093/ jac/dkn515 75. Martinez-Gomariz M, Perumal P, Mekala S, Nombela C, Chaffin WL, Gil C (2009) Proteomic analysis of cytoplasmic and surface proteins from yeast cells, hyphae, and biofilms of Candida albicans. Proteomics 9(8):2230–2252. doi:10.1002/ pmic.200700594 76. Seneviratne CJ, Wang Y, Jin L, Abiko Y, Samaranayake LP (2010) Proteomics of drug resistance in Candida glabrata biofilms. Proteomics 10(7):1444–1454. doi:10.1002/ pmic.200900611 77. Planchon S, Chambon C, Desvaux M, Chafsey I, Leroy S, Talon R, Hebraud M (2007) Proteomic analysis of cell envelope from Staphylococcus xylosus C2a, a coagulase-negative staphylococcus. J Proteome Res 6(9):3566–3580. doi:10.1021/pr070139+ 78. Malen H, Berven FS, Fladmark KE, Wiker HG (2007) Comprehensive analysis of exported proteins from Mycobacte‑ rium tuberculosis H37Rv. Proteomics 7(10):1702–1718. doi:10.1002/pmic.200600853 79. Hansmeier N, Chao TC, Kalinowski J, Puhler A, Tauch A (2006) Mapping and comprehensive analysis of the extracellular

13

Med Microbiol Immunol and cell surface proteome of the human pathogen Corynebac‑ terium diphtheriae. Proteomics 6(8):2465–2476. doi:10.1002/ pmic.200500360 80. Khemiri A, Galland A, Vaudry D, Chan Tchi Song P, Vaudry H, Jouenne T, Cosette P (2008) Outer-membrane proteomic maps and surface-exposed proteins of Legionella pneumophila using cellular fractionation and fluorescent labelling. Anal Bioanal Chem 390(7):1861–1871. doi:10.1007/s00216-008-1923-1 81. Calvo E, Pucciarelli MG, Bierne H, Cossart P, Albar JP, Garcia-Del Portillo F (2005) Analysis of the Listeria cell wall proteome by two-dimensional nanoliquid chromatography coupled to mass spectrometry. Proteomics 5(2):433–443. doi:10.1002/ pmic.200400936 82. Cao Y, Bazemore-Walker CR (2013) Proteomic profiling of the surface-exposed cell envelope proteins of Caulobacter crescen‑ tus. J Proteomics. doi:10.1016/j.jprot.2013.08.011 83. Solis N, Parker BL, Kwong SM, Robinson G, Firth N, Cordwell SJ (2014) Staphylococcus aureus surface proteins involved in adaptation to oxacillin identified using a novel cell shaving approach. J Proteome Res. doi:10.1021/pr500107p 84. Solis N, Parker BL, Kwong SM, Robinson G, Firth N, Cordwell SJ (2014) Staphylococcus aureus surface proteins involved in adaptation to oxacillin identified using a novel cell shaving approach. J Proteome Res 13(6):2954–2972. doi:10.1021/pr500107p 85. Serra DO, Lucking G, Weiland F, Schulz S, Gorg A, Yantorno OM, Ehling-Schulz M (2008) Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis. Proteomics 8(23–24):4995–5010. doi:10.1002/pmic.200800218 86. Sochorova Z, Petrackova D, Sitarova B, Buriankova K, Bezouskova S, Benada O, Kofronova O, Janecek J, Halada P, Weiser J (2014) Morphological and proteomic analysis of early stage air-liquid interface biofilm formation in Mycobacterium smegmatis. Microbiology 160(Pt 7):1346–1356. doi:10.1099/ mic.0.076174-0 87. Guttenplan SB, Kearns DB (2013) Regulation of flagellar motility during biofilm formation. FEMS Microbiol Rev 37(6):849– 871. doi:10.1111/1574-6976.12018 88. Sauer K, Camper AK (2001) Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth. J Bacteriol 183(22):6579–6589. doi:10.1128/ JB.183.22.6579-6589.2001 89. Fletcher JM, Nair SP, Ward JM, Henderson B, Wilson M (2001) Analysis of the effect of changing environmental conditions on the expression patterns of exported surface-associated proteins of the oral pathogen Actinobacillus actinomycetemcomitans. Microb Pathog 30(6):359–368. doi:10.1006/mpat.2000.0439 90. Ang CS, Veith PD, Dashper SG, Reynolds EC (2008) Application of 16O/18O reverse proteolytic labeling to determine the effect of biofilm culture on the cell envelope proteome of Porphyromonas gingivalis W50. Proteomics 8(8):1645–1660. doi:10.1002/pmic.200700557 91. Planchon S, Desvaux M, Chafsey I, Chambon C, Leroy S, Hebraud M, Talon R (2009) Comparative subproteome analyses of planktonic and sessile Staphylococcus xylosus C2a: new insight in cell physiology of a coagulase-negative Staphylococcus in biofilm. J Proteome Res 8(4):1797–1809. doi:10.1021/ pr8004056 92. Guegan C, Garderes J, Le Pennec G, Gaillard F, Fay F, Linossier I, Herry JM, Fontaine MN, Rehel KV (2014) Alteration of bacterial adhesion induced by the substrate stiffness. Colloids Surf B Biointerfaces 114:193–200. doi:10.1016/j. colsurfb.2013.10.010 93. Gilmore JM, Washburn MP (2010) Advances in shotgun proteomics and the analysis of membrane proteomes. J Proteomics 73(11):2078–2091. doi:10.1016/j.jprot.2010.08.005

Med Microbiol Immunol 94. Dunne WM Jr (2002) Bacterial adhesion: Seen any good biofilms lately? Clin Microbiol Rev 15(2):155–166 95. Neu TR, Lawrence JR (2015) Innovative techniques, sensors, and approaches for imaging biofilms at different scales. Trends Microbiol. doi:10.1016/j.tim.2014.12.010 96. Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG (2002) Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 184(4):1140–1154 97. Vilain S, Cosette P, Zimmerlin I, Dupont JP, Junter GA, Jouenne T (2004) Biofilm proteome: Homogeneity or versatility? J Proteome Res 3(1):132–136 98. Allegrucci M, Hu FZ, Shen K, Hayes J, Ehrlich GD, Post JC, Sauer K (2006) Phenotypic characterization of Streptococcus pneumoniae biofilm development. J Bacteriol 188(7):2325– 2335. doi:10.1128/JB.188.7.2325-2335.2006 99. Asakura H, Yamasaki M, Yamamoto S, Igimi S (2007) Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni. FEMS Microbiol Lett 275(2):278–285. doi:10.1111/j.1574-6968.2007.00893.x 100. Hinsa SM, Espinosa-Urgel M, Ramos JL, O’Toole GA (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(4):905–918. doi:10.1046/j.1365-2958.2003.03615.x 101. Petrova OE, Sauer K (2010) The novel two-component regulatory system BfiSR regulates biofilm development by controlling the small RNA rsmZ through CafA. J Bacteriol 192(20):5275– 5288. doi:10.1128/JB.00387-10 102. Deng DM, Buijs MJ, Ten Cate JM (2004) The effects of substratum on the pH response of Streptococcus mutans biofilms and on the susceptibility to 0.2% chlorhexidine. Eur J Oral Sci 112(1):42–47. doi:10.1111/j.0909-8836.2004.00100.x 103. Chaturongkasumrit Y, Takahashi H, Keeratipibul S, Kuda T, Kimura B (2011) The effect of polyesterurethane belt surface roughness on Listeria monocytogenes biofilm formation and its cleaning efficiency. Food Control 22(12):1893–1899. doi:10.1016/j.foodcont.2011.04.032 104. Schlisselberg DB, Yaron S (2013) The effects of stainless steel finish on Salmonella Typhimurium attachment, biofilm formation and sensitivity to chlorine. Food Microbiol 35(1):65–72. doi:10.1016/j.fm.2013.02.005 105. Gallaher TK, Wu S, Webster P, Aguilera R (2006) Identification of biofilm proteins in non-typeable Haemophilus influenzae. BMC Microbiol 6:65. doi:10.1186/1471-2180-6-65 106. Zijnge V, Kieselbach T, Oscarsson J (2012) Proteomics of protein secretion by Aggregatibacter actinomycetemcomitans. PLoS ONE 7(7):e41662. doi:10.1371/journal.pone.0041662 107. Yonezawa H, Osaki T, Kurata S, Fukuda M, Kawakami H, Ochiai K, Hanawa T, Kamiya S (2009) Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm formation. BMC Microbiol 9:197. doi:10.1186/1471-2180-9-197 108. Yamaguchi M, Sato K, Yukitake H, Noiri Y, Ebisu S, Nakayama K (2010) A Porphyromonas gingivalis mutant defective in a putative glycosyltransferase exhibits defective biosynthesis of the polysaccharide portions of lipopolysaccharide, decreased gingipain activities, strong autoaggregation, and increased biofilm formation. Infect Immun 78(9):3801–3812. doi:10.1128/IAI.00071-10 109. Schooling SR, Beveridge TJ (2006) Membrane vesicles: an overlooked component of the matrices of biofilms. J Bacteriol 188(16):5945–5957. doi:10.1128/JB.00257-06 110. Tashiro Y, Uchiyama H, Nomura N (2012) Multifunctional membrane vesicles in Pseudomonas aeruginosa. Environ Microbiol 14(6):1349–1362. doi:10.1111/j.1462-2920.2011.02632.x 111. Altindis E, Fu Y, Mekalanos JJ (2014) Proteomic analysis of Vibrio cholerae outer membrane vesicles. Proc Natl Acad Sci USA. doi:10.1073/pnas.1403683111

112. Schaar V, Nordstrom T, Morgelin M, Riesbeck K (2011) Moraxella catarrhalis outer membrane vesicles carry betalactamase and promote survival of Streptococcus pneumoniae and Haemophilus influenzae by inactivating amoxicillin. Antimicrob Agents Chemother 55(8):3845–3853. doi:10.1128/ AAC.01772-10 113. Bauman SJ, Kuehn MJ (2009) Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells. BMC Microbiol 9:26. doi:10.1186/1471-2180-9-26 114. Yonezawa H, Osaki T, Woo T, Kurata S, Zaman C, Hojo F, Hanawa T, Kato S, Kamiya S (2011) Analysis of outer membrane vesicle protein involved in biofilm formation of Heli‑ cobacter pylori. Anaerobe 17(6):388–390. doi:10.1016/j. anaerobe.2011.03.020 115. McDougald D, Rice SA, Barraud N, Steinberg PD, Kjelleberg S (2012) Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal. Nat Rev Microbiol 10(1):39–50. doi:10.1038/nrmicro2695 116. Webb JS, Thompson LS, James S, Charlton T, Tolker-Nielsen T, Koch B, Givskov M, Kjelleberg S (2003) Cell death in Pseudomonas aeruginosa biofilm development. J Bacteriol 185(15):4585–4592 117. Jackson DW, Suzuki K, Oakford L, Simecka JW, Hart ME, Romeo T (2002) Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli. J Bacteriol 184(1):290–301 118. Huynh TT, McDougald D, Klebensberger J, Al Qarni B, Barraud N, Rice SA, Kjelleberg S, Schleheck D (2012) Glucose starvation-induced dispersal of Pseudomonas aeruginosa biofilms is cAMP and energy dependent. PLoS ONE 7(8):e42874. doi:10.1371/journal.pone.0042874 119. Vaysse PJ, Prat L, Mangenot S, Cruveiller S, Goulas P, Grimaud R (2009) Proteomic analysis of Marinobacter hydro‑ carbonoclasticus SP17 biofilm formation at the alkane-water interface reveals novel proteins and cellular processes involved in hexadecane assimilation. Res Microbiol 160(10):829–837. doi:10.1016/j.resmic.2009.09.010 120. Vaysse PJ, Sivadon P, Goulas P, Grimaud R (2011) Cells dispersed from Marinobacter hydrocarbonoclasticus SP17 biofilm exhibit a specific protein profile associated with a higher ability to reinitiate biofilm development at the hexadecane-water interface. Environ Microbiol 13(3):737–746. doi:10.1111/j.1462-2920.2010.02377.x 121. Solano C, Echeverz M, Lasa I (2014) Biofilm dispersion and quorum sensing. Curr Opin Microbiol 18C:96–104. doi:10.1016/j.mib.2014.02.008 122. Chambers JR, Sauer K (2013) Small RNAs and their role in biofilm formation. Trends Microbiol 21(1):39–49. doi:10.1016/j. tim.2012.10.008 123. Ram RJ, Verberkmoes NC, Thelen MP, Tyson GW, Baker BJ, Blake RC II, Shah M, Hettich RL, Banfield JF (2005) Community proteomics of a natural microbial biofilm. Science 308(5730):1915–1920. doi:10.1126/science.1109070 124. Kolmeder CA, de Been M, Nikkila J, Ritamo I, Matto J, Valmu L, Salojarvi J, Palva A, Salonen A, de Vos WM (2012) Comparative metaproteomics and diversity analysis of human intestinal microbiota testifies for its temporal stability and expression of core functions. PLoS ONE 7(1):e29913. doi:10.1371/journal. pone.0029913 125. Wang HB, Zhang ZX, Li H, He HB, Fang CX, Zhang AJ, Li QS, Chen RS, Guo XK, Lin HF, Wu LK, Lin S, Chen T, Lin RY, Peng XX, Lin WX (2011) Characterization of metaproteomics in crop rhizospheric soil. J Proteome Res 10(3):932–940. doi:10.1021/pr100981r 126. Hall EK, Besemer K, Kohl L, Preiler C, Riedel K, Schneider T, Wanek W, Battin TJ (2012) Effects of resource chemistry on the

13

composition and function of stream hyporheic biofilms. Front Microbiol 3:35. doi:10.3389/fmicb.2012.00035 127. Park C, Helm RF (2008) Application of metaproteomic analysis for studying extracellular polymeric substances (EPS) in activated sludge flocs and their fate in sludge digestion. Water Sci Technol 57(12):2009–2015. doi:10.2166/wst.2008.620 128. Ritter A, Com E, Bazire A, Goncalves MDS, Delage L, Le Pennec G, Pineau C, Dreanno C, Compere C, Dufour A (2012) Proteomic studies highlight outer-membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41. Proteomics 12(21):3180–3192. doi:10.1002/pmic.201100644 129. Provenzano JC, Siqueira JF Jr, Rocas IN, Domingues RR, Paes Leme AF, Silva MR (2013) Metaproteome analysis of endodontic infections in association with different clinical conditions. PLoS ONE 8(10):e76108. doi:10.1371/journal.pone.0076108 130. Burmolle M, Webb JS, Rao D, Hansen LH, Sorensen SJ, Kjelleberg S (2006) Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms. Appl Environ Microbiol 72(6):3916–3923. doi:10.1128/AEM.03022-05 131. Zainal-Abidin Z, Veith PD, Dashper SG, Zhu Y, Catmull DV, Chen YY, Heryanto DC, Chen D, Pyke JS, Tan K, Mitchell HL, Reynolds EC (2012) Differential proteomic analysis of a polymicrobial biofilm. J Proteome Res 11(9):4449–4464. doi:10.1021/pr300201c 132. Kjelleberg S, Molin S (2002) Is there a role for quorum sensing signals in bacterial biofilms? Curr Opin Microbiol 5(3):254– 258. doi:10.1016/S1369-5274(02)00325-9 133. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280(5361):295–298 134. Rutherford ST, Bassler BL (2012) Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med. doi:10.1101/cshperspect.a012427 135. Pearson JP, Pesci EC, Iglewski BH (1997) Roles of Pseu‑ domonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179(18):5756–5767 136. Sakuragi Y, Kolter R (2007) Quorum-sensing regulation of the biofilm matrix genes (pel) of Pseudomonas aeruginosa. J Bacteriol 189(14):5383–5386. doi:10.1128/JB.00137-07 137. Oosthuizen MC, Steyn B, Theron J, Cosette P, Lindsay D, Von Holy A, Brozel VS (2002) Proteomic analysis reveals differential protein expression by Bacillus cereus during biofilm formation. Appl Environ Microbiol 68(6):2770–2780 138. Secor PR, Jennings LK, James GA, Kirker KR, Pulcini ED, McInnerney K, Gerlach R, Livinghouse T, Hilmer JK, Bothner B, Fleckman P, Olerud JE, Stewart PS (2012) Phevalin (aureusimine B) production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression. PLoS ONE 7(7):e40973. doi:10.1371/journal.pone.0040973 139. Iwase T, Uehara Y, Shinji H, Tajima A, Seo H, Takada K, Agata T, Mizunoe Y (2010) Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. Nature 465(7296):346–349. doi:10.1038/nature09074 140. Sugimoto S, Iwamoto T, Takada K, Okuda K, Tajima A, Iwase T, Mizunoe Y (2013) Staphylococcus epidermidis Esp degrades specific proteins associated with Staphylococcus aureus biofilm formation and host-pathogen interaction. J Bacteriol 195(8):1645–1655. doi:10.1128/JB.01672-12 141. De Vriendt K, Theunissen S, Carpentier W, De Smet L, Devreese B, Van Beeumen J (2005) Proteomics of Shewanella oneidensis MR-1 biofilm reveals differentially expressed proteins, including AggA and RibB. Proteomics 5(5):1308–1316. doi:10.1002/pmic.200400989

13

Med Microbiol Immunol 142. Vallenet D, Nordmann P, Barbe V, Poirel L, Mangenot S, Bataille E, Dossat C, Gas S, Kreimeyer A, Lenoble P, Oztas S, Poulain J, Segurens B, Robert C, Abergel C, Claverie JM, Raoult D, Medigue C, Weissenbach J, Cruveiller S (2008) Comparative analysis of Acinetobacters: three genomes for three lifestyles. PLoS ONE 3(3):e1805. doi:10.1371/journal. pone.0001805 143. Lassek C, Burghartz M, Chaves-Moreno D, Otto A, Hentschker C, Fuchs S, Bernhardt J, Jauregui R, Neubauer R, Becher D, Pieper DH, Jahn M, Jahn D, Riedel K (2015) A metaproteomics approach to elucidate host and pathogen protein expression during catheter-associated urinary tract infections. Mol Cell Proteomics. doi:10.1074/mcp.M114.043463 144. Shin JH, Lee HW, Kim SM, Kim J (2009) Proteomic analysis of Acinetobacter baumannii in biofilm and planktonic growth mode. J Microbiol 47(6):728–735. doi:10.1007/ s12275-009-0158-y 145. Marti S, Nait Chabane Y, Alexandre S, Coquet L, Vila J, Jouenne T, De E (2011) Growth of Acinetobacter baumannii in pellicle enhanced the expression of potential virulence factors. PLoS ONE 6(10):e26030. doi:10.1371/journal.pone.0026030 146. Srivastava D, Waters CM (2012) A tangled web: regulatory connections between quorum sensing and cyclic Di-GMP. J Bacteriol 194(17):4485–4493. doi:10.1128/JB.00379-12 147. Romling U, Galperin MY, Gomelsky M (2013) Cyclic diGMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77(1):1–52. doi:10.1128/ MMBR.00043-12 148. Merritt JH, Ha DG, Cowles KN, Lu W, Morales DK, Rabinowitz J, Gitai Z, O’Toole GA (2010) Specific control of Pseu‑ domonas aeruginosa surface-associated behaviors by two c-diGMP diguanylate cyclases. MBio. doi:10.1128/mBio.00183-10 149. Duvel J, Bertinetti D, Moller S, Schwede F, Morr M, Wissing J, Radamm L, Zimmermann B, Genieser HG, Jansch L, Herberg FW, Haussler S (2012) A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aer‑ uginosa. J Microbiol Methods 88(2):229–236. doi:10.1016/j. mimet.2011.11.015 150. Chua SL, Tan SY, Rybtke MT, Chen Y, Rice SA, Kjelleberg S, Tolker-Nielsen T, Yang L, Givskov M (2013) Bis-(3′–5′)cyclic dimeric GMP regulates antimicrobial peptide resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 57(5):2066–2075. doi:10.1128/AAC.02499-12 151. Otto K, Silhavy TJ (2002) Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc Natl Acad Sci USA 99(4):2287–2292. doi:10.1073/ pnas.042521699 152. Stelzer S, Egan S, Larsen MR, Bartlett DH, Kjelleberg S (2006) Unravelling the role of the ToxR-like transcriptional regulator WmpR in the marine antifouling bacterium Pseudoalteromonas tunicata. Microbiology 152(Pt 5):1385–1394. doi:10.1099/ mic.0.28740-0 153. Jager S, Jonas B, Pfanzelt D, Horstkotte MA, Rohde H, Mack D, Knobloch JK (2009) Regulation of biofilm formation by sigma B is a common mechanism in Staphylococcus epidermidis and is not mediated by transcriptional regulation of sarA. Int J Artif Organs 32(9):584–591 154. Saldias MS, Lamothe J, Wu R, Valvano MA (2008) Burkholde‑ ria cenocepacia requires the RpoN sigma factor for biofilm formation and intracellular trafficking within macrophages. Infect Immun 76(3):1059–1067. doi:10.1128/IAI.01167-07 155. Gicquel G, Bouffartigues E, Bains M, Oxaran V, Rosay T, Lesouhaitier O, Connil N, Bazire A, Maillot O, Benard M, Cornelis P, Hancock RE, Dufour A, Feuilloley MG, Orange N, Deziel E, Chevalier S (2013) The extra-cytoplasmic function sigma factor sigX modulates biofilm and virulence-related

Med Microbiol Immunol properties in Pseudomonas aeruginosa. PLoS ONE 8(11):e80407. doi:10.1371/journal.pone.0080407 156. Schembri MA, Kjærgaard K, Klemm P (2003) Global gene expression in Escherichia coli biofilms. Mol Microbiol 48(1):253–267. doi:10.1046/j.1365-2958.2003.03432.x 157. Whiteley M, Bangera MG, Bumgarner RE, Parsek MR, Teitzel GM, Lory S, Greenberg EP (2001) Gene expression in Pseu‑ domonas aeruginosa biofilms. Nature 413(6858):860–864. doi:10.1038/35101627 158. Thompson LS, Webb JS, Rice SA, Kjelleberg S (2003) The alternative sigma factor RpoN regulates the quorum sensing gene rhlI in Pseudomonas aeruginosa. FEMS Microbiol Lett 220(2):187–195. doi:10.1016/S0378-1097(03)00097-1 159. Khemiri A, Lecheheb SA, Chi Song PC, Jouenne T, Cosette P (2014) Proteomic regulation during Legionella pneumoph‑ ila biofilm development: decrease of virulence factors and enhancement of response to oxidative stress. J Water Health 12(2):242–253. doi:10.2166/wh.2014.103 160. Iyer VS, Hancock LE (2012) Deletion of sigma(54) (rpoN) alters the rate of autolysis and biofilm formation in Entero‑ coccus faecalis. J Bacteriol 194(2):368–375. doi:10.1128/ JB.06046-11 161. Hao B, Mo ZL, Xiao P, Pan HJ, Lan X, Li GY (2013) Role of alternative sigma factor 54 (RpoN) from Vibrio anguillarum M3 in protease secretion, exopolysaccharide production, biofilm formation, and virulence. Appl Microbiol Biotechnol 97(6):2575–2585. doi:10.1007/s00253-012-4372-x 162. Clark ME, He Z, Redding AM, Joachimiak MP, Keasling JD, Zhou JZ, Arkin AP, Mukhopadhyay A, Fields MW (2012) Transcriptomic and proteomic analyses of Desulfo‑ vibrio vulgaris biofilms: carbon and energy flow contribute to the distinct biofilm growth state. BMC Genom 13:138. doi:10.1186/1471-2164-13-138 163. Dharmaprakash A, Mutt E, Jaleel A, Ramanathan S, Thomas S (2014) Proteome profile of a pandemic Vibrio parahaemo‑ lyticus SC192 strain in the planktonic and biofilm condition. Biofouling 30(6):729–739. doi:10.1080/08927014.2014.91669 6 164. Mace C, Seyer D, Chemani C, Cosette P, Di-Martino P, Guery B, Filloux A, Fontaine M, Molle V, Junter GA, Jouenne T (2008) Identification of biofilm-associated cluster (bac) in Pseudomonas aeruginosa involved in biofilm formation and virulence. PLoS ONE 3(12):e3897. doi:10.1371/journal. pone.0003897 165. Vera M, Krok B, Bellenberg S, Sand W, Poetsch A (2013) Shotgun proteomics study of early biofilm formation process of Aci‑ dithiobacillus ferrooxidans ATCC 23270 on pyrite. Proteomics 13(7):1133–1144. doi:10.1002/pmic.201200386 166. Koerdt A, Orell A, Pham TK, Mukherjee J, Wlodkowski A, Karunakaran E, Biggs CA, Wright PC, Albers SV (2011) Macromolecular fingerprinting of Sulfolobus species in biofilm: a transcriptomic and proteomic approach combined with spectroscopic analysis. J Proteome Res 10(9):4105–4119. doi:10.1021/ pr2003006 167. Marceau M, Nassif X (1999) Role of glycosylation at Ser63 in production of soluble pilin in pathogenic Neisseria. J Bacteriol 181(2):656–661 168. Sampathkumar B, Napper S, Carrillo CD, Willson P, Taboada E, Nash JH, Potter AA, Babiuk LA, Allan BJ (2006) Transcriptional and translational expression patterns associated with immobilized growth of Campylobacter jejuni. Microbiology 152(Pt 2):567–577. doi:10.1099/mic.0.28405-0 169. Grantcharova N, Peters V, Monteiro C, Zakikhany K, Romling U (2010) Bistable expression of CsgD in biofilm development of Salmonella enterica serovar typhimurium. J Bacteriol 192(2):456–466. doi:10.1128/JB.01826-08

170. Irie Y, Starkey M, Edwards AN, Wozniak DJ, Romeo T, Parsek MR (2010) Pseudomonas aeruginosa biofilm matrix polysaccharide Psl is regulated transcriptionally by RpoS and posttranscriptionally by RsmA. Mol Microbiol 78(1):158–172. doi:10.1111/j.1365-2958.2010.07320.x 171. Singer SW, Erickson BK, VerBerkmoes NC, Hwang M, Shah MB, Hettich RL, Banfield JF, Thelen MP (2010) Posttranslational modification and sequence variation of redox-active proteins correlate with biofilm life cycle in natural microbial communities. ISME J 4(11):1398–1409. doi:10.1038/ismej.2010.64 172. Derouiche A, Cousin C, Mijakovic I (2012) Protein phosphorylation from the perspective of systems biology. Curr Opin Biotechnol 23(4):585–590. doi:10.1016/j.copbio.2011.11.008 173. Kishi M, Hasegawa Y, Nagano K, Nakamura H, Murakami Y, Yoshimura F (2012) Identification and characterization of novel glycoproteins involved in growth and biofilm formation by Por‑ phyromonas gingivalis. Mol Oral Microbiol 27(6):458–470. doi:10.1111/j.2041-1014.2012.00659.x 174. Sakamoto A, Terui Y, Yamamoto T, Kasahara T, Nakamura M, Tomitori H, Yamamoto K, Ishihama A, Michael AJ, Igarashi K, Kashiwagi K (2012) Enhanced biofilm formation and/or cell viability by polyamines through stimulation of response regulators UvrY and CpxR in the two-component signal transducing systems, and ribosome recycling factor. Int J Biochem Cell Biol 44(11):1877–1886. doi:10.1016/j.biocel.2012.07.010 175. Morris AR, Visick KL (2013) The response regulator SypE controls biofilm formation and colonization through phosphorylation of the syp-encoded regulator SypA in Vibrio fischeri. Mol Microbiol 87(3):509–525. doi:10.1111/mmi.12109 176. Ribet D, Cossart P (2010) Post-translational modifications in host cells during bacterial infection. FEBS Lett 584(13):2748– 2758. doi:10.1016/j.febslet.2010.05.012 177. Wu WL, Liao JH, Lin GH, Lin MH, Chang YC, Liang SY, Yang FL, Khoo KH, Wu SH (2013) Phosphoproteomic analysis reveals the effects of PilF phosphorylation on type IV pilus and biofilm formation in Thermus thermophilus HB27. Mol Cell Proteomics 12(10):2701–2713. doi:10.1074/mcp.M113.029330 178. Petrova OE, Sauer K (2009) A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. PLoS Pathog 5(11):e1000668. doi:10.1371/journal. ppat.1000668 179. Mikkelsen H, Sivaneson M, Filloux A (2011) Key two-component regulatory systems that control biofilm formation in Pseu‑ domonas aeruginosa. Environ Microbiol 13(7):1666–1681. doi:10.1111/j.1462-2920.2011.02495.x 180. Hancock LE, Perego M (2004) The Enterococcus faecalis fsr two-component system controls biofilm development through production of gelatinase. J Bacteriol 186(17):5629–5639. doi:10.1128/JB.186.17.5629-5639.2004 181. Mikkelsen H, Sivaneson M, Filloux A (2011) Key two-component regulatory systems that control biofilm formation in Pseu‑ domonas aeruginosa. Environ Microbiol 13(7):1666–1681. doi:10.1111/j.1462-2920.2011.02495.x 182. Posch G, Pabst M, Brecker L, Altmann F, Messner P, Schaffer C (2011) Characterization and scope of S-layer protein O-glycosylation in Tannerella forsythia. J Biol Chem 286(44):38714– 38724. doi:10.1074/jbc.M111.284893 183. Hitchen PG, Twigger K, Valiente E, Langdon RH, Wren BW, Dell A (2010) Glycoproteomics: a powerful tool for characterizing the diverse glycoforms of bacterial pilins and flagellins. Biochem Soc Trans 38(5):1307–1313. doi:10.1042/BST0381307 184. Scott NE, Parker BL, Connolly AM, Paulech J, Edwards AV, Crossett B, Falconer L, Kolarich D, Djordjevic SP, Hojrup P, Packer NH, Larsen MR, Cordwell SJ (2011) Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy

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collisional dissociation, and electron transfer dissociation MS applied to the N-linked glycoproteome of Campylobac‑ ter jejuni. Mol Cell Proteomics 10(2):M000031–MCP000201. doi:10.1074/mcp.M000031-MCP201 185. Hitchen PG, Dell A (2006) Bacterial glycoproteomics. Microbiology 152(Pt 6):1575–1580. doi:10.1099/mic.0.28859-0 186. Besanceney-Webler C, Jiang H, Wang W, Baughn AD, Wu P (2011) Metabolic labeling of fucosylated glycoproteins in Bac‑ teroidales species. Bioorg Med Chem Lett 21(17):4989–4992. doi:10.1016/j.bmcl.2011.05.038 187. Magalhaes A, Ismail MN, Reis CA (2010) Sweet receptors mediate the adhesion of the gastric pathogen Helicobacter pylori: glycoproteomic strategies. Expert Rev Proteomics 7(3):307–310. doi:10.1586/epr.10.18 188. Gerlach JQ, Kilcoyne M, Farrell MP, Kane M, Joshi L (2012) Differential release of high mannose structural isoforms by fungal and bacterial endo-beta-N-acetylglucosaminidases. Mol BioSyst 8(5):1472–1481. doi:10.1039/c2mb05455h 189. Benz I, Schmidt MA (2002) Never say never again: protein glycosylation in pathogenic bacteria. Mol Microbiol 45(2):267– 276. doi:10.1046/j.1365-2958.2002.03030.x 190. Roth J (2002) Protein N-glycosylation along the secretory pathway: relationship to organelle topography and function, protein quality control, and cell interactions. Chem Rev 102(2):285– 303. doi:10.1021/cr000423j 191. Khemiri A, Naudin B, Franck X, Chan Tchi Song P, Jouenne T, Cosette P (2013) N-Glycosidase treatment with (18)O labeling and de novo sequencing argues for flagellin FliC glycopolymorphism in Pseudomonas aeruginosa. Anal Bioanal Chem 405(30):9835–9842. doi:10.1007/s00216-013-7424-x 192. McNamara M, Tzeng SC, Maier C, Zhang L, Bermudez LE (2012) Surface proteome of “Mycobacterium avium subsp. hominissuis” during the early stages of macrophage infection. Infect Immun 80(5):1868–1880. doi:10.1128/IAI.06151-11 193. Jarrell KF, Jones GM, Kandiba L, Nair DB, Eichler J (2010) S-layer glycoproteins and flagellins: reporters of archaeal posttranslational modifications. Archaea. doi:10.1155/2010/612948 194. Banerjee A, Ghosh SK (2003) The role of pilin glycan in neisserial pathogenesis. Mol Cell Biochem 253(1–2):179–190 195. Jennings MP, Jen FE, Roddam LF, Apicella MA, Edwards JL (2011) Neisseria gonorrhoeae pilin glycan contributes to CR3 activation during challenge of primary cervical epithelial cells. Cell Microbiol 13(6):885–896. doi:10.1111/j.1462-5822.2011.01586.x 196. Yin QY, de Groot PW, de Koster CG, Klis FM (2008) Mass spectrometry-based proteomics of fungal wall glycoproteins. Trends Microbiol 16(1):20–26. doi:10.1016/j.tim.2007.10.011 197. Taff HT, Nett JE, Zarnowski R, Ross KM, Sanchez H, Cain MT, Hamaker J, Mitchell AP, Andes DR (2012) A Candida biofilminduced pathway for matrix glucan delivery: implications for drug resistance. PLoS Pathog 8(8):e1002848. doi:10.1371/journal.ppat.1002848 198. Babauta J, Renslow R, Lewandowski Z, Beyenal H (2012) Electrochemically active biofilms: facts and fiction. A review. Biofouling 28(8):789–812. doi:10.1080/08927014.2012.71032 4 199. Kalathil S, Khan MM, Lee J, Cho MH (2013) Production of bioelectricity, bio-hydrogen, high value chemicals and bioinspired nanomaterials by electrochemically active biofilms. Biotechnol Adv 31(6):915–924. doi:10.1016/j.biotechadv.2013.05.001 200. Biffinger JC, Byrd JN, Dudley BL, Ringeisen BR (2008) Oxygen exposure promotes fuel diversity for Shewanella oneiden‑ sis microbial fuel cells. Biosens Bioelectron 23(6):820–826. doi:10.1016/j.bios.2007.08.021 201. Lindblad P, Lindberg P, Oliveira P, Stensjo K, Heidorn T (2012) Design, engineering, and construction of photosynthetic

13

Med Microbiol Immunol microbial cell factories for renewable solar fuel production. Ambio 41(Suppl 2):163–168. doi:10.1007/s13280-012-0274-5 202. Pereira-Medrano AG, Knighton M, Fowler GJ, Ler ZY, Pham TK, Ow SY, Free A, Ward B, Wright PC (2013) Quantitative proteomic analysis of the exoelectrogenic bacterium Arcobacter butzleri ED-1 reveals increased abundance of a flagellin protein under anaerobic growth on an insoluble electrode. J Proteomics 78:197–210. doi:10.1016/j.jprot.2012.09.039 203. Sun G, Thygesen A, Ale MT, Mensah M, Poulsen FW, Meyer AS (2014) The significance of the initiation process parameters and reactor design for maximizing the efficiency of microbial fuel cells. Appl Microbiol Biotechnol. doi:10.1007/ s00253-013-5486-5 204. Monroe D (2007) Looking for chinks in the armor of bacterial biofilms. PLoS Biol 5(11):e307. doi:10.1371/journal. pbio.0050307 205. Perrot F, Hebraud M, Charlionet R, Junter GA, Jouenne T (2001) Cell immobilization induces changes in the protein response of Escherichia coli K-12 to a cold shock. Electrophoresis 22(10):2110–2119. doi:10.1002/15222683(200106)22:103.0.CO;2-B 206. Schmidt I, Steenbakkers PJ, op den Camp HJ, Schmidt K, Jetten MS (2004) Physiologic and proteomic evidence for a role of nitric oxide in biofilm formation by Nitrosomonas europaea and other ammonia oxidizers. J Bacteriol 186(9):2781–2788 207. Benard L, Litzler PY, Cosette P, Lemeland JF, Jouenne T, Junter GA (2009) Proteomic analysis of Staphylococcus aureus biofilms grown in vitro on mechanical heart valve leaflets. J Biomed Mater Res A 88(4):1069–1078. doi:10.1002/ jbm.a.31941 208. Zimaro T, Thomas L, Marondedze C, Garavaglia BS, Gehring C, Ottado J, Gottig N (2013) Insights into xanthomonas axonopodis pv. citri biofilm through proteomics. BMC Microbiol 13:186. doi:10.1186/1471-2180-13-186 209. Coquet L, Cosette P, De E, Galas L, Vaudry H, Rihouey C, Lerouge P, Junter GA, Jouenne T (2005) Immobilization induces alterations in the outer membrane protein pattern of Yers‑ inia ruckeri. J Proteome Res 4(6):1988–1998. doi:10.1021/ pr050165c 210. Meyer DH, Fives-Taylor PM (1994) Characteristics of adherence of Actinobacillus actinomycetemcomitans to epithelial cells. Infect Immun 62(3):928–935 211. Siroy A, Cosette P, Seyer D, Lemaitre-Guillier C, Vallenet D, Van Dorsselaer A, Boyer-Mariotte S, Jouenne T, De E (2006) Global comparison of the membrane subproteomes between a multidrug-resistant Acinetobacter baumannii strain and a reference strain. J Proteome Res 5(12):3385–3398. doi:10.1021/ pr060372s 212. Danese PN, Pratt LA, Kolter R (2000) Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J Bacteriol 182(12):3593–3596 213. Rodrigues DF, Elimelech M (2009) Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation. Biofouling 25(5):401–411. doi:10.1080/08927010902833443 214. Chapman MR, Robinson LS, Pinkner JS, Roth R, Heuser J, Hammar M, Normark S, Hultgren SJ (2002) Role of Escheri‑ chia coli curli operons in directing amyloid fiber formation. Science 295(5556):851–855. doi:10.1126/science.1067484 215. Boucher JC, Yu H, Mudd MH, Deretic V (1997) Mucoid Pseudomonas aeruginosa in cystic fibrosis: characterization of muc mutations in clinical isolates and analysis of clearance in a mouse model of respiratory infection. Infect Immun 65(9):3838–3846 216. Boucher JC, Schurr MJ, Yu H, Rowen DW, Deretic V (1997) Pseudomonas aeruginosa in cystic fibrosis: role of mucC in the

Med Microbiol Immunol regulation of alginate production and stress sensitivity. Microbiology 143(Pt 11):3473–3480 217. Davey ME, Caiazza NC, O’Toole GA (2003) Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol 185(3):1027–1036 218. Pham TK, Roy S, Noirel J, Douglas I, Wright PC, Stafford GP (2010) A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen Tannerella forsythia. Proteomics 10(17):3130–3141. doi:10.1002/pmic.200900448 219. Shibata S, Visick KL (2012) Sensor kinase RscS induces the production of antigenically distinct outer membrane vesicles that depend on the symbiosis polysaccharide locus in Vibrio fis‑ cheri. J Bacteriol 194(1):185–194. doi:10.1128/JB.05926-11 220. Brady RA, Leid JG, Camper AK, Costerton JW, Shirtliff ME (2006) Identification of Staphylococcus aureus proteins recognized by the antibody-mediated immune response to a

biofilm infection. Infect Immun 74(6):3415–3426. doi:10.1128/ IAI.00392-06 221. Vadyvaloo V, Otto M (2005) Molecular genetics of Staphylo‑ coccus epidermidis biofilms on indwelling medical devices. Int J Artif Organs 28(11):1069–1078 222. Romero D, Aguilar C, Losick R, Kolter R (2010) Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Proc Natl Acad Sci USA 107(5):2230–2234. doi:10.1073/ pnas.0910560107 223. Renier S, Chambon C, Viala D, Chagnot C, Hebraud M, Desvaux M (2013) Exoproteomic analysis of the SecA2-dependent secretion in Listeria monocytogenes EGD-e. J Proteomics 80C:183–195. doi:10.1016/j.jprot.2012.11.027

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