Xanthomonas axonopodis pv. citri (X. a. pv. citri) is a gram-negative plant pathogenic bacteria that causes citrus canker [1]. This phytopathogen invades host plant tissues entering through stomata or wounds and then colonizes the apoplast of fruits, foliage and young stems and symptoms of infection appear as raised corky lesions. At the final stage, plant tissue epidermis is broken due to cell hyperplasia, which allows bacterial dispersal to other plants by windblown rain. Persistent and severe disease can lead to defoliation, dieback and fruit drop thereby reducing yields, and hence causing serious economic losses. Moreover, quarantine restrictions are imposed to producing areas with citrus canker thus hampering commercialization of fresh fruit [2].

In most environments, bacteria primarily grow in association with surfaces, leading to the formation of biofilms. These biofilms generally consist of microbial cells attached to a surface and covered with an extracellular matrix composed of protein and polysaccharides [3]. The elevated population density forming a biofilm can increase biological processes that single cells cannot perform. Specifically, the biofilm lifestyle can offer increased protection against environmental stresses and increase bacterial resistance against host defense responses and antimicrobial tolerance. Biofilms also allow for consortial metabolism and may increase the possibility for horizontal gene transfer [3]. For most pathogenic bacteria, attachment to surfaces and successive biofilm formation are essential steps in the development of chronic infections and maintenance on host tissues [4]. In plant pathogens, biofilm formation also allows for increased bacterial cell density that in turn helps to achieve a critical mass of cells at a specific location to initiate and sustain interactions with host plants [5].

X. a. pv. citri biofilm formation appears to be a common feature during infection and different X. a. pv. citri mutants impaired in surface attachment, aggregation and hence in biofilm formation are also deficient in pathogenesis [6–8]. The lack of exopolysaccharide (EPS), the main component of the matrix surrounding biofilm cells, reduces epiphytic survival in planta[9] and has a negative impact on X. a. pv. citri virulence [10–14]. Other mutant strains affected in lipopolysaccharide (LPS) or glucan biosynthesis are impaired in the formation of structured biofilms and show reduced virulence symptoms [15–17]. Moreover, the two-component regulatory system ColR/ColS, which plays a major role in the regulation of X. a. pv. citri pathogenicity, also modulates biofilm formation [18]. In this context, further insight into X. a. pv. citri biofilm formation was gained by screening X. a. pv. citri transposon insertion mutants for biofilm-defective phenotypes, leading to the identification of several genes related to X. a. pv. citri biofilm formation [19]. Given that for X. a. pv. citri too, biofilm formation is a requirement to achieve maximal virulence, we have used proteomics to identify differentially expressed proteins with a view to gain further insight into the process of biofilm formation.

Phenotypic analysis of X. a. pv. citri biofilm development

Biofilm formation generally requires a number of different processes including the initial surface attachment of cells, cell multiplication to form micro-colonies and maturation of the biofilm [20]. For a better understanding of the dynamics of this process in X. a. pv. citri, biofilm structure of a GFP-expressing X. a. pv. citri strain (Xac-GFP) was observed at different growth stages by confocal laser scanning microscopy. To this end, Xac-GFP was cultured in static liquid XVM2 medium, a minimal medium that mimics the nutritional conditions found in plant tissues [21]. As previously described, biofilms are important for X. a. pv. citri virulence, and thus XVM2 medium was used to analyze bacterial biofilm formation in a plant-like environment. After one day of growth, some cells began to attach to the surface of the PVC plate wells, however, the majority of cells remained dispersed in the culture medium (Figure 1). After three days of growth, cells initiated accumulation and formation of a biofilm (Figure 1), and after seven days, Xac-GFP cells formed a distinctly structured and dense biofilm consisting of large cell aggregations separated by a network of large channels (Figure 1) that ensured appropriate micronutrient and oxygen fluxes [22]. We also evaluated the population size of these biofilms and observed that at day seven of growth the biofilms reached a maximum population size of 1 x 10⁹ cfu/ml. In a planktonic culture in XVM2 medium, a similar maximal population size is reached in early stationary phase. Therefore, these two conditions of growth were used to identify differentially expressed proteins between the two lifestyles at their respective maximum population sizes and prior to the occurrence of noticeable cell death.

Two-dimensional gel electrophoretic analysis of protein expression and mass spectrometric identification of the X. a. pv. citri biofilm proteome

Since proteomics is a powerful method to obtain systems information on the physiology of bacterial cells, we aimed at analyzing and characterizing mature biofilms of X. a. pv. citri, and compare the proteome to that of planktonic X. a. pv. citri cells. Total proteins of these cultures were extracted and separated by two-dimensional gel electrophoresis (2-DE) (see “Methods” section). Protein extractions were performed from three independent biological samples, and two technical replicate gels for each cell type were compared. A total of 46 protein spots were differentially regulated (Figure 2), excised and processed for analysis by mass spectrometry. Forty-one spots were identified, corresponding to a total of 53 proteins (Additional file 1: Table S1), while five spots (spots 250, 382, 348, 352 and 357) remained unidentified, probably due to the very low protein concentration in these spots. Some protein spots were assigned to more than one protein, possibly because the proteins co-migrated as a result of having the same pI and molecular weight. This pattern of co-migration is not uncommon in proteomic studies and was reported previously [23, 24]. The 31 up- and 22 down-regulated X. a. pv. citri biofilm proteins were classified into different categories based on their functions [25] (Additional file 1: Table S1). The protein spot displaying the strongest up-regulation was 50S ribosomal protein L4 (XAC0973; +5.1 fold; spot 79), followed by TonB-dependent receptor (XAC3489; +4.9 fold; spot 168), while the protein spot with the most pronounced down-regulation was an ATP synthase beta chain (XAC3649; -10.7 fold; spot 76). Here we focus on interpreting a subset (see Table 1) of the differentially expressed biofilm proteins.

Functional characterization of differentially regulated X. a. pv. citri biofilm proteins

The identified differentially expressed proteins were used to determine enriched GO categories in biological processes, molecular function and cellular localization. The main enriched categories for the up- and down-regulated proteins with an average fold change of minimum ±1.5 are represented graphically (Figure 3). The major biological processes and cellular localization categories that changed in the X. a. pv. citri biofilms are ‘transporter activity’ and ‘external encapsulating structure’, respectively. The categories that showed enrichment in the up-regulated proteins include ‘catabolic process’, ‘external encapsulating structure’, ‘receptor activity’ and ‘transporter activity’; while most of the down-regulated proteins were in the categories of ‘biosynthetic process’, ‘nucleobase, nucleoside, nucleotide and nucleic acid metabolic process’, ‘metabolic process’, ‘catabolic process’ and ‘generation of precursor metabolites and energy’.

Specifically for X. a. pv. citri, we observed only a small overlap with recently published data that identified genes involved in biofilm formation by transposon mutagenesis [19]. The common proteins include UDP-glucose dehydrogenase and a TonB-dependent receptor proteins [19]. A possible explanation for this may be that transposon mutagenesis also identifies genes that are indirectly involved in biofilm formation, and additionally many of the identified genes may be required for the first stages of biofilm formation, such as adherence to the surface. Here, we focused on the proteins present in mature biofilms and for this reason many of the genes found in the genome-wide scale assay may be not differently expressed in this structure.

The most enriched categories for the up-regulated proteins in X. a. pv. citri biofilm are ‘external encapsulating structure’, ‘transporter activity’ and ‘receptor activity’, and include the outer membrane receptors termed TonB-dependent receptors (TBDRs). Among them, the OmpA-related protein (XAC4274, spot 103) and TonB-dependent receptors (XAC3050, spots 1, 2, 74, 219; XAC3071, spot 466 and XAC3489, spots 55 and 168) were up-regulated, while the TonB-dependent receptors (XAC3168, spot 38 and XAC3444, spot 15) were down-regulated in X. a. pv. citri biofilms. The TBDR proteins are localized in the outer membrane of gram-negative bacteria and their most prominent recognized role is the transport of iron-siderophore complexes and cobalamin into the periplasm [34]. Transport via TBDRs is an active process requiring energy that is provided by the inner membrane TonB-ExbB-ExbD protein complex [35]. Generally, expression of the genes encoding for these receptors is activated under conditions of iron starvation and repressed in the presence of iron by the ferric-uptake regulator (Fur) repressor [36]. Several genome sequences of gram-negative bacteria were examined to determine the number of TBDRs present in each genome, and it was demonstrated that only a number of these bacteria, among them the Xanthomonas species, have an over-representation of TBDRs [37]. Most of the analyzed bacteria with an elevated number of TBDRs share the ability to metabolize complex carbohydrates. Therefore, it was postulated that some Xanthomonas TBDRs might be involved in the transport of plant-derived molecules [37], and this hypothesis was confirmed with the characterization of two TBDRs from Xanthomonas campestris pv. campestris and Caulobacter crescentus, that transport sucrose and maltodextrins, respectively [37, 38]. It was also suggested that other TBDRs might be involved in signal transduction processes [39]. Our proteomics results suggest that TBDRs participates in X. a. pv. citri biofilm formation and probably has a role in the adaptation to this lifestyle by modulating carbohydrate transport and utilization or signal perception and/or transduction. This hypothesis is supported by a recent study in X. a. pv. citri that showed that a transposon insertion mutant in a different TBDR (XAC0144) resulted in impaired in biofilm formation [19].

Other proteins that were up-regulated in biofilms and belonging to the categories ‘transporter activity’ and ‘receptor activity’ processes were identified as outer membrane proteins (OMPs) or porins. Porins are integral membrane β-barrel proteins and act as a selective barrier enabling the passage of nutrients, waste products, water and chemical signals through formed pores [40]. Within the class of porins, FadL (XAC0019, spot 609), a protein that allows the passage of fatty acids [41], was up-regulated in X. a. pv. citri biofilms, and was previously observed as important for bacterial virulence [14]. In Pseudomonas fluorescens, FadL has been reported in biofilms on abiotic surfaces, and it has been suggested that the long chain of fatty acids bound to FadL alter surface hydrophobicity and therefore adhesion characteristics [27]. Interestingly, the outer membrane porin termed “Regulator of pathogenicity factors” (RpfN) in the X. a. pv. citri genome (XAC2504, spots 151, 429, 486, 526, 555) was also up-regulated in the biofilms. This particular porin is encoded in a genomic region along with genes specialized in internalization of fructose and was suggested to play a role in carbohydrate transport [42], that in turn may be necessary for X. a. pv. citri adaptation to biofilm lifestyle. Moreover, the Burkholderia pseudomallei homolog to RpfN, named OprB, was shown to be important for optimal biofilm formation [43]. The OmpW (XAC3664; spot 432) was another up-regulated porin in X. a. pv. citri biofilms. It is involved in the transport of small hydrophilic molecules across the bacterial outer membrane [44]. Recent studies in Salmonella typhimurium suggest that this porin may have a role in the protection of bacteria against various forms of environmental stress by operating as efflux channel for toxic compounds [45]. We therefore hypothesize that OmpW may be involved in protecting X. a. pv. citri biofilms.

UDP-glucose dehydrogenase (UGD) (XAC3581, spot 220) was over-expressed in X. a. pv. citri biofilms (Table 1) and enriched in the category ‘metabolic process’. This enzyme catalyzes the conversion of UDP-glucose to UDP-glucuronic acid and the cellular functions of UGD have been investigated in a number of organisms establishing a role in detoxification, polysaccharide biosynthesis as well as embryonic development [46]. Moreover, a double mutant in Pseudomonas aeruginosa UGD (PA2022-PA3559) produced thinner biofilms than the wild type PAO1 and it has been suggested that the functional role of UGD in P. aeruginosa, involves hyaluronic acid (polysaccharide consisting of alternative GlcUA and GlcNAc residues) synthesis, which also contributes to biofilm formation [47]. In X. campestris pv. campestris, UGD is required for the biosynthesis of xanthan gum that is a component of the exopolysaccharide (EPS). Therefore, together with the well established role of X. a. pv. citri EPS in bacterial adherence and biofilm formation [10, 11, 19], the over-expression of UGD in X. a. pv. citri biofilms is consistent with a major role of EPS under biofilm growth conditions. Also consistent with this conclusion is the absence of biofilm formation in a X. a. pv. citri UGD deletion mutant [19].

The non-fimbrial adhesin, YapH (XAC2151, spot 86), a protein up-regulated in X. a. pv. citri biofilms, is an adhesin that belongs to the family of the filamentous hemagglutinins secreted by the two-partner secretion system [48]. In X. axonopodis pv. phaseoli, a YapH ortholog was discovered to be involved in the adhesion process to biotic and abiotic surfaces and also in biofilm formation [26]. We previously characterized another filamentous hemagglutinin named X. a. pv. citri FhaB, and showed that it is critical for X. a. pv. citri biofilm formation [6]. In agreement with these studies, the present results substantiate the role of this family of adhesins in X. a. pv. citri biofilm formation.

Among the category ‘nucleic acid metabolic process’, the polynucleotide phosphorylase (PNPase) (XAC2683, spot 153) was down-regulated in biofilms. PNPase is an important enzyme involved in RNA processing and turnover [49]. Recently, it was demonstrated that PNPase negatively regulates cell aggregation and biofilm formation in E. coli by inhibiting the expression of genes involved in the production of the EPS poly-N-acetylglucosamine at post-transcriptional level [33]. In this context, our results may suggest that in X. a. pv. citri, this enzyme also enables the adaptation to the biofilm lifestyle.

Several proteins involved in other categories such as protein synthesis, folding and stabilization were up-regulated in X. a. pv. citri biofilm, including the Elongation factor Tu (Ef-Tu) (XAC0957, spots 26, 173), the 50s ribosomal protein L4 (XAC0973; spot 79) and the molecular chaperone DnaK (XAC1522, spot 416). Our results are in agreement with reports which described an increase in 30S ribosomal protein S1, Ef-Tu, 50s ribosomal protein L1, and DnaK during biofilm formation in Streptococcus pneumoniae[29]. Similarly, Pseudomonas aeruginosa biofilms display an up-regulation of ribosome recycling factor and 50S ribosomal protein [50]. The increase in Ef-Tu and the 50s ribosomal protein L4 observed in X. a. pv. citri biofilm may be related to participation in protein synthesis and folding and this in turn may be a specific requirements of the lifestyle. However, for Ef-Tu, other functions such as participation in bacterial aggregation also need to be considered since this factor has also been identified as a cell wall associated component in several bacterial species where it mediates the binding to host proteins (e.g. to human plasminogen in Listeria monocytogenes) [51] and in Mycoplasma pneumoniae where it directly binds fibronectin, suggesting a role of Ef-Tu in aggregation [52]. Ef-Tu was also over-expressed in the wild type strain of Lactobacillus crispatus as compared to an isogenic mutant that lost the aggregative phenotype and strengthening the claim for a role in adhesion [53]. Moreover, in the citrus pathogen Xylella fastidiosa, Ef-Tu was reported to be up-regulated in biofilms [32]. Recent work demonstrated that in X. a. pv. citri, DnaK is necessary for the bacteria to achieve full virulence [14]. Several proteomics reports associate the up-regulation of DnaK to biofilm formation. Among them, a dnaK knock-down mutant of Streptococcus mutans with reduced levels of DnaK (<95%) shows impaired biofilm-forming capacity [30], while DnaK expression was up-regulated in a Prevotella intermedia biofilm-forming strain when compared to a variant lacking biofilm formation [31].

Several proteins that were enriched in the categories ‘metabolic process’, ‘generation of precursor metabolites and energy’, ‘catabolic process’ and ‘biosynthetic process’ showed altered expression patterns in X. a. pv. citri biofilms. A number of enzymes of the tricarboxylic acid (TCA) cycle were also detected as differentially expressed in the biofilm compared to planktonic cultures. Since the TCA cycle plays a central role in metabolism, our finding indicates that the two lifestyles may have markedly different metabolic and energy requirements. The three differentially expressed enzymes of the TCA cycle are citrate synthase (XAC3388, spot 235), malate dehydrogenase (XAC1006, spot 98) and dihydrolipoamide S-succinyltransferase (XAC1534, spot 121). Citrate synthase catalyzes the first reaction in the TCA cycle converting oxaloacetate and acetyl-coenzyme A into citrate and coenzyme A (CoA). Incidentally, it has been observed that a citrate synthase of Burkholderia cenocepacia is necessary for optimum levels of biofilm formation and virulence [28]. In Geobacter sulfurreducens, uniform expression of citrate synthase genes was noted throughout biofilms [54]. The second over-expressed protein in biofilms was identified as malate dehydrogenase, the enzyme that catalyzes the reversible conversion of L-malate to oxaloacetate, and the synthesis of this enzyme is influenced by cell growth conditions such as oxygenation and the nature of carbon substrates [55]. Succinate dehydrogenase (spot 591) was down-regulated in the biofilm. Succinate dehydrogenase complex catalyzes the oxidation of succinate to fumarate, donating FADH₂ for oxidative phosphorylation. In the presence of oxygen, the TCA cycle operates as an oxidative pathway coupled to aerobic respiration. Under oxygen-limiting conditions, the TCA cycle operates as reductive (incomplete) pathway dedicated largely to the synthesis of precursors blocking the steps from α-ketoglutarate to succinyl-CoA. In the reductive pathway, succinyl-CoA is synthesized using the enzyme fumarate reductase as an alternative to succinate dehydrogenase, leading to the down-regulation of succinate dehydrogenase [56]. Given the down-regulation of succinate dehydrogenase in X. a. pv. citri biofilms, our results might suggest that in biofilms, the TCA cycle is converted into the reductive pathway possibly because of oxygen limitations under these growth conditions. Accordingly, succinate dehydrogenase is directly linked to the respiratory chain and in E. coli, an increase in oxygen respiration correlated with succinate dehydrogenase over-expression [57]. Moreover, X. a. pv. citri biofilms showed a down-regulation of protein involved in energy generation, such as ATP-synthase (XAC3649, spot 76), phosphoglycerate kinase (XAC3347, spot 442) and NADH-ubiquinone oxidoreductase (XAC2699, spot 422). Phosphoglycerate kinase enzyme is involved in later reactions of the glycolytic pathway; therefore its inhibition should lead to an increased pool of glycolytic intermediates in the early steps that might benefit biosynthetic processes. Furthermore, an enzyme involved in cellular energy homeostasis, adenylate kinase (XAC3437, spot 49) was also down-regulated in X. a. pv. citri biofilms. This enzyme catalyzes the interconversion of adenine nucleotides generating ATP and AMP, a metabolic signaling molecule.

Several antioxidant enzymes enriched in the ‘metabolic process’ are involved in secondary metabolism. In bacteria, the normal course of aerobic metabolism produces reactive oxygen species (ROS) with the concomitant requirement for the constitutive expression of ROS scavenging systems such as antioxidant enzymes [58]. In accordance with our hypothesis that X. a. pv. citri biofilms have a reduced aerobic respiration rate, these biofilms showed a down-regulation in antioxidant enzymes like a NAD(PH) nitroreductase (XAC0554, spot 60), a short chain dehydrogenase (XAC1484, spot 434) and an aerobic coproporphyinogen-III oxidase (XAC4109, spot 533). Nitroreductases are a family of evolutionarily related proteins involved in the reduction of nitrogen-containing compounds and the short-chain dehydrogenases/reductases represent a large family of enzymes, most of which are NAD- or NADP-dependent oxidoreductases [59]. Coproporphyrinogen III oxidase is encoded by the hemF gene that is involved in heme-biosynthesis [60]. This protein was shown to be a member of the hydrogen peroxide (oxidative stress)-induced regulon responsible for protecting cells from oxidative damage [61]. The 4-hydroxy-2-oxoglutarate (KHG)/phospho-2-dehydro-3-deoxygluconate (KDPG) aldolases (XAC2067, spot 331), a key enzyme for the Entner-Doudoroff pathway, was down-regulated in biofilms. The KHG aldolase catalyzes the interconversion of 4-hydroxy-2-oxoglutarate into pyruvate and glyoxylate in the glyoxylate cycle, while KDPG-aldolase induces the interconversion of 6-phospho-2-dehydro-3-deoxy-D-gluconate into pyruvate and glyceraldehyde 3-phosphate and the two enzymes are structurally and functionally related [62]. One primary function of the glyoxylate cycle is to replenish the tricarboxylic and dicarboxylic acid intermediates that are normally provided by the TCA cycle. In X. a. pv. citri biofilms, several enzymes of the TCA cycle are up-regulated suggesting a reduced requirement for the glyoxylate cycle under this static growth condition.

One GO category (‘signal transduction’) is enriched in down-regulated proteins only and comprises a putative two-component system sensor histidine kinase under-expressed in X. a. pv. citri biofilms (XAC1991, spot 420). Previously, it was shown that a X. a. pv. citri mutant that has a transposon insertion at the intergenic region between XAC1990 and XAC1991 induces milder infection symptoms than the wild type strain [14]. Since these genes have the same genomic orientation, this mutation probably impairs only XAC1991 expression. These data may suggest that besides its involvement in X. a. pv. citri pathogenicity, this sensor histidine kinase may also be involved in the adaptation to different lifestyles.

Transcriptional analysis of selected genes encoding differentially expressed proteins

We selected some of these genes for further validation by quantitative real-time PCR (qRT-PCR). Total RNA was extracted from X. a. pv. citri mature biofilms and from planktonic cells, both grown as for the proteomic study. Bacterial cDNA was obtained from 1 μg of total RNA in both growth conditions. The assay was performed with specific primers for the following X. a. pv. citri genes: XAC3581 (UDP-glucose dehydrogenase), XAC0973 (50S ribosomal protein L4), XAC0957 (EfTu), XAC2504 (RpfN), XAC3489 (TonB-dependent receptor), XAC2151 (YapH), XAC3664 (OmpW) and XAC1522 (DnaK). We noted that the changes in transcript levels of theses genes mirrored the changes observed in the proteomics analysis (p < 0.05) (Figure 4).

Several lines of evidence indicate that X. a. pv. citri biofilm formation plays an important part in bacterial pathogenicity. Among them, studies on a variety of impaired biofilm forming mutants have revealed the importance of this lifestyle for the citrus pathogen. Here we identified proteins differentially expressed in a mature X. a. pv. citri biofilm as compared to free planktonic cultured cells. A larger number of differentially expressed proteins are down-regulated and relate to metabolic processes suggesting a reduction in energy requirements. Further experiments will show to which extent this is due to reduced metabolic activity at this growth stage. It is also noteworthy, firstly, that proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the GO terms ‘generation of precursor metabolites and energy’ and secondly, that the biofilm proteome mainly displayed changes in outer membrane and receptor or transport proteins suggesting that they may have a role in maintaining a functional external structure as well as enabling appropriate flow of molecules and signals required in this lifestyle. This study is the first report of a X. a. pv. citri biofilm proteome and the information gained will support future comparative analyses of differentially expressed genes and/or proteins involved in biofilm formation. In addition, the data will also inform approaches to a more detailed physiological investigation into the function of individual proteins and their role in biofilm formation.