Pectobacterium carotovorum subsp. carotovorum [formerly E. carotovora subsp. carotovora (Pcc])] PC1 [formerly EC1 (wild type)], its derivative strains, ΔcytR (aflagellated and non-motile mutant), ΔfliC (non-motile and aflagellated mutant), flhD::Tn5 (aflagellated and non-motile mutant) and ΔmotA (flagellated and non-motile mutant) and D. dadantii (formerly Erwinia chrysanthemi) 3937 (wild type) used in this study has been described earlier by Matsumoto et al. (2003), Haque and Tsuyumu (2005), and Hossain et al. (2005). The strains were freshly grown in yeast extract peptone (YP) medium (1% peptone, 0.5% yeast extract, pH 6.8) at 27°C. Nalidixic acid (30 μg/mL) and kanamycin (50 μg/mL) were added to the media when required, and the optical density (OD) of the culture was measured by a spectrophotometer (Intertech, Inc. Tokyo, Japan) at 660 nm.

Yeast extract peptone, Luria-Bertani (LB) medium (1% of tryptone, 0.5% of yeast extract, 0.5% of NaCl, pH 7.0), Salt-optimized broth (SOB) plus 2% of glycerol (SOBG) medium (per liter: 20 g of tryptone, 5 g of yeast extract, 0.5 g of NaCl, 0.186 g of KCl, 2.4 g of MgSO_4.7H₂O and 2% of glycerol), King’s B (KB) medium (per liter: 10 g of peptone, 1.5 g of K₂HPO₄, l5 g of MgSO_4.7H₂O and 15 mL of glycerol), yeast peptone dextrose adenine (YPDA) medium (per liter: 20 g of yeast extract, 40 g of peptone, 40 g of glucose monohydrate, and 80 mg of adenine hemisulfate) and M63 glycerol minimal medium [per liter, 2.5 g of NaCl, 3 g of KH₂PO₄, 7 g of K₂HPO₄, 2 g of (NH₄)₂SO₄, 0.5 mg of FeSO₄, 2 g of thiamine hydrochloride, and 2 g of glycerol] were used. In order to quantify the impacts of media and temperature on AL biofilm formation, a single colony of the Pcc PC1 and the ΔcytR mutant was grown in shake (180 rpm) culture in YP broth at 27°C until early stationary phase (OD₆₆₀ at 1.0). Afterward, 50 μl of each culture [ca. 10⁷ colony forming unit (CFU)/mL] were suspended in glass test tubes containing 5 mL of the broth. Two separate suspensions were made and incubated at two different temperatures (27 and 37°C, respectively) for 72 h in static condition. In order to study the role of carbon source on AL biofilm formation, 2% glycerol in SOBG was replaced by 2% of glucose, sucrose, or mannitol. Among the media, the Pcc PC1 produced a thick and robust AL biofilm on SOBG broth at 27°C. The SOBG media and 27°C temperature was therefore taken to study the AL biofilm unless otherwise noted in this manuscript.

Bacterial strains formed fragile to rigid AL biofilm on SOBG broth. In order to quantify the rigid biomass, samples were prepared as follows: 72 h-old AL biofilms were gently transferred to the fresh test tubes containing 2 mL of distilled water and were vortexed with sterile glass beads. The amount of the biomass in the rigid AL biofilms (OD₆₀₀) were quantified using an UV spectrophotometer (Ultrospec 3000, Pharmacia Biotech, Cambridge, England). In case of fragile AL biofilm, 1 mL of planktonic culture was carefully collected by the pipette and OD₆₀₀ was measured. Afterward, each fragile AL biofilm was mixed with 2 mL of planktonic culture and was vortexed. The OD₆₀₀ of planktonic culture was then subtracted from the OD₆₀₀ of the planktonic culture plus biomass of the fragile AL biofilm. This would provide the amount of fragile biomass present in the AL biofilm.

The SAL biofilms formed at 37°C were also quantified as described in Haque et al. (2012). In brief, the suspension was carefully removed after 72 h and washed the glass test tubes with distilled water. Afterward, 0.05% (w/v) crystal violet was added and incubated for 30 min followed by rinsing with distilled water. Bound crystal violet was eluted using 95% ethanol and the SAL biofilm was quantified (OD₅₇₀) using UV spectrophotometer (Ultrospec 3000, Pharmacia Biotech, Cambridge, England).

Congo red and Calcofluor binding assays were carried out as described in Haque et al. (2009, 2012) with a few modifications. Initially, each bacterial strain (Pcc PC1, ΔcytR, ΔfliC, flhD::Tn5, ΔmotA, and D. dadatii 3937) [used as positive control for the expression of red, dry and rough (rdar) phenotype and expression of cellulose] was grown in YP broth overnight at 27°C under shaking condition (180 rpm). Each bacterial culture was serial diluted 1:100 [ca. 10⁵ CFU/mL] and 2 μL of culture were spotted (four spot in each plate) onto SOBG agar plates containing 40 μg/mL of Congo red (Sigma-Aldrich, St. Louis, MO, United States) or 200 μg/mL of Calcofluor white (Sigma-Aldrich, St. Louis, MO, United States). The plates were then incubated at 27°C in static condition, and photographs were taken after 48 h (for Congo red binding). On the other hand, after 48 h incubation, the plates were placed under UV light (366 nm) and photographs were taken for Calcofluor binding.

Amount of cellulose produced in the matrix of the AL biofilms of the bacterial strains (Pcc PC1 ΔcytR, ΔfliC, flhD::Tn5, and ΔmotA) were quantified as described in Anriany et al. (2006) with a few modifications. In brief, strains were grown at 27°C in SOBG broth for 3 days in static condition. Then 3 g (wet weight) of AL biofilm masses were gently collected with sterile spatula and were dried by freeze dryer (Eyela, Freeze dryer, FDU-830, Japan). The dry masses were mixed with 4.5 mL of acetic-nitric reagent (8:2:1 acetic acid: nitric acid: distilled water) and boiled for 20 min. The boiled mixture was then centrifuged and the supernatant was discarded. The pellet was transferred to a Corex centrifuged bottles, washed twice with sterile distilled water and dried in a clean bench. The dried pellet was mixed with 150 μL of concentrated H₂SO₄ with gentle shaking for 1 h at 27°C. The amount of cellulose was determined by adding 750 μL anthrone (Sigma-Aldrich, St. Louis, MO, United States) reagent (0.2 g in 100 mL H₂SO₄). The Avicel cellulose (Sigma-Aldrich, St. Louis, MO, United States) was used as standard this case, and the cellulose were quantified at 620 nm.

Wild type (Pcc PC1) and ΔcytR mutant were grown in SOBG broth until AL biofilm was formed. Total RNA was extracted thereafter from the associated bacteria using an RNA isolation kit (Qiagen, Hilden, Germany) and was subjected to DNase I treatment with the TURBO DNase kit (Ambion, Inc., United States) as instructed by the manufacturer. The purity and concentration of RNA was estimated using a Nanodrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States).

Primers (Table 1) were designed based on Pcc PC1 DNA sequences that can be retrieved from the following address¹. cDNA synthesis, verification of the efficiencies of the primers and PCR were carried out as described in Haque et al. (2015). The relative values of transcriptional level were calculated using the ΔΔC_T method. The abundance of specific gene was initially normalized using 16S rRNA which was shown to be invariant using best keeper (Matsumoto et al., 2003). The relative expression ratio was calculated as the differences between the cycle threshold (C_T) values and was determined using the following equation:

Fold change = 2^-ΔΔC_T, where ΔΔC_T for gene j = (C_T,J – C_T,16SrRNA)_mutant – (C_T,J – C_T,16SrRNA)_wildtype.

Attachment assays were performed as described in Jahn et al. (2008) with a few modifications. In brief, radish (Raphanus sativus) seed (10 g) was collected from Horticulture Research Centre of Bangladesh Agricultural Research Institute, Gazipur, Bangladesh. Seeds were surface sterilized with 75 mL of 1.5% sodium hypochloride for 30 min on a rotary shaker at 200 rpm and was followed by several washes using sterile distilled water. Single colonies of bacterial strain were inoculated into SOBG broth and were grown overnight at 27°C with agitation. It was then centrifuged before each strain was resuspended in sterile distilled water. Approximately 10⁴ CFU/mL bacteria were used. For attachment assays, seeds were germinated in sterile water for 4 days with regular water changes. The sprouts were transferred in sterile 50 mL conical tubes (10 sprouts/tube) and were incubated in bacterial suspension for 4 h at 27°C with gentle shaking (50 rpm). Sprouts were then rinsed three times with sterile distilled water and homogenized. After serial dilution, the homogenate was plated and incubated at 27°C for 24 h for colonies to be enumerated. The experiment was repeated at least three times with at least seven sprouts analyzed per strain each time.

Stationary (OD₆₆₀ at 1.5) phase cells (grown in shaking condition) and cells-associated with AL biofilm of the wild type and the ΔcytR mutant were prepared as described in Haque et al. (2012). In order to prepare the planktonic cells (i.e., cells under the AL biofilm), 1 mL of planktonic culture was carefully collected after 72 h incubation followed by centrifugation. The pellet was then re-suspended in sterile distilled water. In order to test the sensitivities, the stationary-phase, the planktonic cells and the AL biofilm cells (ca. 10⁸ CFU/mL) of the wild type and the ΔcytR mutant were separately exposed to acidic pH 4.0 (40 mM citric acid and 20 mM dibasic sodium phosphate, pH 4.0) and oxidative stress (H₂O₂ at 10 mM) which corresponds to a lethal concentration (Haque et al., 2009). The suspensions were incubated for a further 2 h at 27°C. The number of CFU was determined by serial dilution and plating onto SOBG agar just prior to inoculation and during every 30 min over a 2-h incubation. The survival of treated cells was normalized to the number of CFU at the beginning of the test.

All the experiments were conducted in complete randomized design with three replications and repeated at least three times unless otherwise stated. Data were analyzed using Student’s t-test of the SAS software system 8.02 (SAS Institute, Cary, NC, United States).

In order to quantify the impacts of media composition and temperature on AL biofilm formation, the Pcc PC1 and the ΔcytR mutant cells (ca. 10⁷ CFU/mL) were suspended in five different media (SOBG or KB or LB or YP or M63 glycerol minimal media) and was incubated at two different temperatures (27 or 37°C) in static condition. Selection of five different media and two different temperatures was important to generate information that previous researches did not shed light on. A study by Hossain and Tsuyumu (2006) showed that Pcc PC1 produces SAL biofilm on YP broth plus salts of M63 minimal medium in the wells of microtiter plates made of PVC at 27°C in static condition. They also examined two other media such as YP and LB broth but neither of them produced any SAL or AL biofilms in Pcc PC1. The present study particularly deals with AL biofilm formation in glass test tubes under different environmental conditions which were not examined in any other contemporary researchers dealing with Pcc PC1 and its ΔcytR mutant. This study aimed to contribute toward this area of concern, as it was carried out under different media and temperature for a comprehensive result. The results of this study showed that Pcc PC1 formed AL biofilm on SOBG, KB, and YPDA broth after 72 h incubation only at 27°C (Figure 1A). In fact, the AL biofilm in SOBG, KB, and YPDA broth did not continue to grow after 7 days (data not shown). However, the AL biofilm in SOBG broth showed signs of detachment at day 7 (data not shown). The AL biofilms formed by Pcc PC1 on the SOBG broth was quite rigid and was denser compared to those on the KB and YPDA broth. On the other hand, biofilms on YPDA and KB by Pcc PC1 were fragile and hence, were dispersed when the samples were disturbed. AL biofilms also acquired a sizable biomass when the glycerol in SOBG was replaced by glucose, sucrose, and mannitol (data not shown). The ΔcytR mutant, on the other hand, produced thinner, lighter and more fragile AL biofilm on SOBG, YPDA and KB broth (Figure 1A). Quantitative analysis of the biomass at OD₆₀₀ showed that the wild type produced significantly (P < 0.001) higher biofilm biomass in SOBG (4.18-fold), YPDA (3.18-fold), and KB (3.10-fold) broth compared to the ΔcytR mutant at 27°C (Figure 1B). At 37°C however, the ΔcytR mutant and its wild counterpart formed only SAL biofilms on SOBG, YPDA, and KB broth (Figure 1A). The Pcc PC1 produced a prominent SAL biofilm on SOBG broth compared to those on the YPDA and KB broth (Figure 1A). The other three media (YP, LB, and M63 glycerol minimal) did not produce any SAL or AL biofilm for both of these strains under any of the temperatures (27 or 37°C) (data not shown). Similar to the AL biofilms, the amount of SAL biofilm (OD₅₇₀) was also significantly (P < 0.001) higher in the wild type on SOBG (2.4-fold) and YPDA (1.9-fold) compared to the ΔcytR mutant (Figure 1B). Initially however, both wild type (Pcc PC1) and ΔcytR mutant cells grew faster in all the broths except in M63 glycerol minimal medium where the growth was slightly delayed both at 27°C (Figures 1C,D) and at 37°C (Figures 1E,F). In general, the cell growth was slightly delayed in ΔcytR mutant compared to that of the wild type in YP broth at both temperatures (Figures 1C–F). These results suggested that biofilm formation controlled by the CytR homolog of Pcc PC1 is regulated by media composition and temperature. Bacterial growth might play an important role in this case, although it is very unlikely to be amongst the major determinants.

Divalent cations such as Mg²⁺, Ca²⁺, Cu²⁺, Mn²⁺, and Zn²⁺ have been shown to be the inducers and stabilizers of the biofilm formation processes (Song and Leff, 2006; Liang et al., 2010; Haque et al., 2012). However, information regarding the role of such cations in the formation of AL biofilms by Pcc PC1 and its ΔcytR mutant was not available in the literature. In this study, the Pcc PC1 (wild type) and ΔcytR mutant cells were inoculated in SOBGMg^– (magnesium deprived) and SOBGMg^– with 0.009 M of Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺ and Zn²⁺, were incubated at 27°C under shaking (180 rpm) condition. The growth rate was not significantly different between the wild type (Figure 2A) and the ΔcytR mutant (data not shown) in different incubation period for any divalent cations. Both the wild type and the mutant cells grew quickly and attained its maximum at 18 h of incubation period both in SOBGMg^– and SOBGMg^– containing 0.009 M Mg²⁺ and Ca²⁺ (Figure 2A). In SOBGMg^– containing 0.009 M Mn²⁺, Cu²⁺, and Zn²⁺ however, the growth was slow and hence, took 24 h to reach the growth maximum (Figure 2A). Interestingly, it was only after 72 h incubation (in static condition) both the strains developed thinner to denser AL biofilm only on SOBGMg^– containing the divalent cations (Figure 2B). Unlike this, the SOBGMg^– broth at 27°C did not show any AL biofilms either in the wild type or in the ΔcytR mutant (Figure 2B). Compared to the ΔcytR mutant, wild type built a denser and robust AL biofilm on SOBGMg^– containing Mg²⁺ and Ca²⁺ and a thinner and more fragile AL biofilm on SOBGMg^– containing Cu²⁺, Mn²⁺, and Zn²⁺ (Figure 2B). When quantified, the production of biomass in the ΔcytR mutant was found to be significantly (P < 0.001) lower than that of its wild counterpart (Figure 2C). These results clearly indicate that the CytR homolog may positively regulate AL biofilm formation in Pcc PC1 responding to divalent cations.

During early stages of plant infection, pathogenic bacteria often confronts an acidic pH that ranges from pH 4.5 to 6.5 (Grignon and Sentenac, 1991). In order to assess whether Pcc PC1 and ΔcytR mutant thrives under acidic conditions, these strains were exposed to acidic pH ranges (Figure 3A) using malic acid on SOBG broth at 27°C. The reason behind using malic acid is to stimulate the acidity in plant apoplast that contains malate (Grignon and Sentenac, 1991). The results showed that none of the strains grew at pH 4.5 while showing slower growth at pH 5.0 and pH 5.5 under shaking condition at 27°C. Bacterial cell growth did not differ significantly between the wild type (Figure 3A) and the mutant (data not shown) at different incubation period. The wild type formed a delicate AL biofilm on SOBG broth at pH 5.0 and pH 5.5 after 120 h in static condition (Figure 3B). On the other hand, ΔcytR mutant cells did not grow at the same condition (Figure 3B). After 72 h incubation at 27°C, the wild type formed a thinner AL biofilm on SOBG broth at pH 6.0 compared to pH 7.0 (Figure 3B). In case of ΔcytR mutant, only a few cells aggregated on the top and did not cover the whole surface at pH 6.0 (Figure 3B). Our results indicated that acidic pH delayed the AL biofilm formation. The compounds and/or the genes responsible for AL biofilm formation may be not produced or expressed in acidic conditions leading to AL biofilm formation of Pcc PC1.

Osmolarity plays a great role in biofilm formation (Solomon et al., 2005; Rinaudi et al., 2006). In order to assess how osmolarity affects the formation of AL biofilm, NaCl and D-sorbitol were used as osmotic agents in the SOBG broth that contains 0.5 g/L NaCl. We however, tested the growth of bacterial cells under different concentrations (0.05, 0.1, 0.2, 0.3 M) of NaCl in shaking condition at 27°C. The results suggest that the growth (OD₆₆₀) of both strains was unaffected due to the changes in osmolarity except for 0.3 M NaCl. Compared to the ΔcytR mutant, a firm and intense AL biofilm was developed by the wild type on the SOBG broth containing 0.05 and 0.1 M NaCl after 72 h incubation. Unlike this, 0.3 M NaCl produced small quantities of cells by the wild type and the mutant in shaking condition (Figure 4A) and consequently, a few cells were aggregated on the surface of the standing culture (Figure 4B). Similar results were also observed when 0.3 M NaCl was replaced by 0.3 M D-sorbitol (data not shown). Conversely, when 0.3 M NaCl/D-sorbitol was replaced by 0.3 M sucrose, both wild type and the ΔcytR mutant cells grew rapidly (Figure 4A). At this condition, wild type produced a stronger and thicker AL biofilm compared to the ΔcytR mutant after 72 h incubation in static condition (Figure 4B). These observations suggest that osmotic stress negatively affect AL biofilm formation due to poor growth of the bacterial cells which CytR homolog of Pcc PC1 is able to regulate. This could possibly include the regulation of expression of other components required for AL biofilm formation.

Higher oxygen concentrations play an important role in AL biofilm formation (Gerstel and Römling, 2001; Liang et al., 2010). However, this general statement needs to be tested and quantified for the Pcc PC1 and its ΔcytR mutant. In order to accomplish this, the wild type and the ΔcytR mutant cells (ca. 10⁷ CFU/mL) were suspended in glass test tubes containing 5 mL SOBG broth. It was then sealed with 1.5 mL of sterile liquid paraffin oil and incubated at 27°C for 7 days in static condition. None of the strains formed an AL biofilm although the turbidity of the cells in the broth was increased (Figure 4C). Similar results were also observed when wild type and the ΔcytR mutant cells (ca. 10⁷ CFU/mL) was suspended in glass test tubes containing 5 mL SOBG broth and incubated at 27°C in anaerobic chamber (Thermo, Inc., Portsmouth, NH, United States) for 7 days (Figure 4C). This could be due to the lack of expression of compounds responsible for AL biofilm formation as stated by Gerstel and Römling (2001).

Flagella-mediated motility was shown to play an important role in AL biofilm formation (Jahn et al., 2008; Yang et al., 2008; Haque et al., 2012). On the other hand, Hossain and Tsuyumu (2006) showed that motility, but not the presence of flagella, is required for SAL biofilm formation in microtiter plates of Pcc PC1. Because CytR homolog positively regulates flagella-mediated motility (Matsumoto et al., 2003), we examined whether CytR homolog of Pcc PC1 also regulates AL biofilm by controlling the motility itself. In order to confirm whether the motility itself or the presence of flagella is required for AL biofilm formation on SOBG broth at 27°C in stationary condition, we used three aflagellated and non-motile mutants such as ΔcytR, ΔfliC and flhD::Tn5, and one flagellated and non-motile mutant such as ΔmotA (essential for flagellar rotation but not required for flagellar assembly) of Pcc PC1. All the mutants produced a lighter and fragile AL biofilm compared to their wild counterpart after 72 h incubation at 27°C (Figure 5A). The results also showed that AL biofilm was not increased in the mutants even after longer incubation period (data not shown). When the biofilm biomass was quantified, wild type (Pcc PC1) was found to have produced significantly (P < 0.001) more (2.9-, 5.5-, 4.5-, and 9.6-fold) AL biofilms than that of cytR, ΔfliC, flhD::Tn5, and ΔmotA, respectively (Figure 5B). These data, therefore concludes that motility itself, but not the presence of flagella, is required for AL biofilm formation which the CytR homolog of Pcc PC1 is able to control.

Biofilm producing bacteria develop red, dry and rough (rdar) phenotype (also known as rugose/wrinkled phenotype) on Congo red agar plates (Römling, 2005; Haque et al., 2012; Milanov et al., 2015). We hypothesized that CytR homolog, FliC, FlhD, and MotA may affect the expression of rdar phenotype. The results of this study show that the expression of rdar phenotype on Congo red agar plates was indistinguishable between the mutants (such as ΔcytR, ΔfliC, flhD::Tn5, and ΔmotA) and the wild type (Figure 5C). Therefore, the rdar phenotype may not be controlled by the CytR homolog, FliC, FlhD, and MotA in Pcc PC1.

It is understood that rdar expressing bacteria usually binds with the cellulose specific dye Calcofluor (Zogaj et al., 2001; Solano et al., 2002; Römling, 2005; Uhlich et al., 2006; Steenackers et al., 2012; Milanov et al., 2015). We therefore evaluated whether wild type Pcc PC1 and the mutants (ΔcytR, ΔfliC, flhD::Tn5, and ΔmotA) also binds to Calcofluor. The results showed that the wild type (Pcc PC1) had induced bright fluorescence similar to D. dadantii 3937, while all the mutants (ΔcytR, ΔfliC, flhD::Tn5, and ΔmotA) weakly induced bright fluorescence (Figure 5D). This result indicated that wild type Pcc PC1 may produce more cellulose-rich EPS compared to the mutants.

Because cellulose production and AL biofilm formation are correlated in bacteria, we quantified cellulose production in the matrix of the biofilms. The wild type was found to have produced more cellulose (49.7 ± 2.3 ng) than the mutants of ΔcytR (21.3 ± 1.7 ng), ΔfliC (13.4 ± 2.3 ng), flhD::Tn5 (24.3 ± 2.7 ng), and ΔmotA (9.8 ± 0.8 ng) mutant. According to these results, the increase in Calcofluor binding seemed to have been reflected in the increase of cellulose production. This indicates that the CytR homolog is capable of regulating the AL biofilm formation by controlling both cellulose production and motility in Pcc PC1.

Bacterial cellulose synthesis (bcs) operons, bcsABZC and bcsEFG, are required for cellulose production leading to AL biofilm formation in E. coli and S. enterica serovar Typhimurium (Römling et al., 2000; Solano et al., 2002; Da Re and Ghigo, 2006). Homology searches revealed that bcsA, bcsB, bcsC, bcsE, bcsF, and bcsG of Pcc PC1 had 67.5, 56.5, 50.5, 41.7, 30, and 62.3 similarity to the translated products of bcsA, bcsB, bcsC, bcsE, bcsF, and bcsG of E. coli K-12, respectively, at the amino acid level. We hypothesized that the CytR homolog may affect expression of bcs genes in Pcc PC1. In order to test this hypothesis, the expression of bcsA (a cellulase synthase) and bcsE (maximal cellulose expresser), was measured by quantitative reverse transcription-PCR using mRNA collected from the AL biofilms formed by the wild type and its ΔcytR mutant. Expressions of bcsA and bcsE were significantly (P < 0.001) reduced (6.3- and 11.1-fold) in the ΔcytR mutant compared to that in the wild type (Figure 6). Therefore, the CytR homolog may positively regulates the transcription of bcs genes in Pcc PC1.

c-di-GMP is catalyzed by the GGDEF domain proteins present in diguanylate cyclases (DGCs) (Hickman et al., 2005; Ryjenkov et al., 2005) and hydrolyzed by either the EAL or HD-GYP domains present in PDEs (Ryan et al., 2006). The AdrA is a GGDEF domain protein that is able to synthesize the c-di-GMP which binds to the BcsA (Kader et al., 2006; Morgan et al., 2014) and BcsE (Fang et al., 2014). The adrA is also required for cellulose biosynthesis in S. enterica serovar Typhimurium (Römling et al., 2000; Zogaj et al., 2001). In D. dadantii 3937, ecpB (a GGDEF-EAL protein) and ecpC (a EAL protein) were shown to be negatively regulated the biofilm formation (Yi et al., 2010). The Pcc PC1 genome encodes 17 GGDEF domain proteins, six EAL domain proteins and three GGDEF-EAL domain proteins¹. In this study, we found that the expression of adrA (a GGDEF domain protein) was considerably lower (3.2-fold) in the ΔcytR mutant (Figure 6) compared to its wild type. However, the expression of PC1_RS16795 (a EAL domain protein) had a dramatic increase (2.7-fold) in the ΔcytR mutant compared to the wild type. Thus, CytR homolog may control the GGDEF (AdrA) and EAL (PC1_RS16795) domain proteins in Pcc PC1.

Because ΔflhD, ΔfliC, and ΔmotA produced significantly the lower amount of cellulose than their wild counterpart, in this study, expressions of flhD, fliA, fliC, and motA were measured by quantitative reverse transcription-PCR using mRNA collected from the AL biofilms formed by the wild type and its ΔcytR mutant. Expressions of flhD (2.3-fold), fliC (11-fold), fliA (8.3-fold), and motA (16.7-fold) were significantly (P < 0.001) decreased in the ΔcytR mutant than the wild type (Figure 6). These results indicated that CytR homolog positively controls the expressions of flhD, fliA, fliC, and motA in Pcc PC1.

In D. dadantii 3937, T3SS regulatory (hrpX, hrpY, hrpS, and hrpL), structural (hrpA, hrcJ) and effector (hrpN) genes are shown to be required for AL biofilm formation (Yap et al., 2005). GGDEF and EAL domain proteins in D. dadantii 3937 were also shown to be negatively regulated both the T3SS and biofilm formation (Yi et al., 2010). In our study, we found that GGDEF and EAL domain proteins are also controlled by the CytR homolog of Pcc PC1 (Figure 6). Thus, the expression of T3SS genes was examined by quantitative reverse transcription-PCR using mRNA collected from AL biofilms of the wild type and ΔcytR mutant. Compared to the wild type, the expression of hrpX (2.8-fold), hrpL (4.3-fold), hrpA (4.8-fold) and hrpN (7.1-fold) was found to have significantly (P < 0.001) reduced in the ΔcytR mutant (Figure 6). Thus, CytR homolog is positively controlled the hrp genes in Pcc PC1. These results also suggested that CytR homolog is required for hrpL expression, which in turn activates the expression of hrp genes in the HrpL regulon in Pcc PC1 (Chatterjee et al., 2002b; Yap et al., 2005; Yi et al., 2010).

In our experiment however, the expression of hrpS did not differ significantly between the wild type and its ΔcytR mutant (Figure 6). CytR homolog may affect the expression of hrpL through the known hrpL regulators, such as RpoN (σ⁵⁴), RsmA (a small RNA-binding protein) or RsmB (a regulatory RNA that binds to and sequesters the negative effect of RsmA on hrpL mRNA by forming RsmA–RsmB complex) (Chatterjee et al., 2002b; Yi et al., 2010). The amount of rsmA and rsmB transcripts in ΔcytR mutant was similar to that in the wild type (Figure 6). Compared to the wild type, the expression of rpoN was significantly (P < 0.001) lower (3.7-fold) in the ΔcytR mutant (Figure 6). Therefore, the CytR homolog may be termed as a regulator of hrpL expression which it achieves by altering the expression of rpoN in Pcc PC1.

Because CytR homolog can regulate AL biofilm formation, we hypothesized that similar genes were also required for attachment to plant tissues as well. The ΔcytR mutant was significantly reduced (P < 0.001) in attachment to radish sprouts compared with the wild type (Figure 7). We also tested the ΔfliC, flhD::Tn5, and ΔmotA mutants for the attachment. Compared to the wild type, their attachment was dramatically reduced (P < 0.001) in these mutants (Figure 7). Thus, CytR homolog, FliC, FlhD, and MotA may regulate both AL biofilm formation in culture and also during attachment to plants in Pcc PC1.

When Pcc PC1 infects a plant, it confronts an unfavorable environment such as acidity and oxidative stresses. These conditions may play a vital role in survival and expression of the virulence factors (Toth et al., 2003) of any organism. Since biofilms play an important role in the survival of bacteria, we compared the sensitivities of AL biofilms and other cells under conditions of acidity (pH 4.0) and oxidative stress generated by 10 mM H₂O₂. Results suggested that the biofilm cells were more resistant than the planktonic and stationary-phase cells (Figure 8). The wild type (Pcc PC1) was found to have shown more resistance compared to the ΔcytR mutant under acidic pH (Figure 8A). All the planktonic- and stationary-phase cells of the ΔcytR mutant were killed by 10 mM H₂O₂ within the first 60 min, and only 9% of the bacteria-associated with AL biofilm of the ΔcytR mutant survived within 60-min exposure time (Figure 8B). On the contrary, a 60-min exposure of stationary-phase, planktonic and AL biofilm cells of the wild type (Pcc PC1) against 10 mM H₂O₂ resulted in 4, 13, and 45% survival, respectively (Figure 8B). Thus, formation of AL biofilm controlled by the CytR homolog may play a great role in the survival of Pcc PC1 under unfavorable environments.