Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803

Abstract

The phototropic bacterium Synechocystis sp. strain PCC 6803 is able to adapt its morphology in order to survive in a wide range of harsh environments. Under conditions of high salinity, planktonic cells formed cell aggregates in culture. Further observations using crystal violet staining, confocal laser scanning microscopy, and field emission-scanning electron microscopy confirmed that these aggregates were Synechocystis biofilms. Polyamines have been implicated in playing a role in biofilm formation, and during salt stress the content of spermidine, the major polyamine in Synechocystis, was reduced. Two putative arginine decarboxylases, Adc1 and Adc2, in Synechocystis were heterologously expressed in Escherichia coli and purified. Adc2 had high arginine decarboxylase activity, whereas Adc1 was much less active. Disruption of the adc genes in Synechocystis resulted in decreased spermidine content and formation of biofilms even under nonstress conditions. Based on the characterization of the adc mutants, Adc2 was the major arginine decarboxylase whose activity led to inhibition of biofilm formation, and Adc1 contributed only minimally to the process of polyamine synthesis. Taken together, in Synechocystis the shift from planktonic lifestyle to biofilm formation was correlated with a decrease in intracellular polyamine content, which is the inverse relationship of what was previously reported in heterotroph bacteria.IMPORTANCE There are many reports concerning biofilm formation in heterotrophic bacteria. In contrast, studies on biofilm formation in cyanobacteria are scarce. Here, we report on the induction of biofilm formation by salt stress in the model phototrophic bacterium Synechocystis sp. strain PCC 6803. Two arginine decarboxylases (Adc1 and Adc2) possess function in the polyamine synthesis pathway. Inactivation of the adc1 and adc2 genes leads to biofilm formation even in the absence of salt. The shift from planktonic culture to biofilm formation is regulated by a decrease in spermidine content in Synechocystis This negative correlation between biofilm formation and polyamine content, which is the opposite of the relationship reported in other bacteria, is important not only in autotrophic but also in heterotrophic bacteria.

Keywords: arginine decarboxylase; biofilm; cyanobacteria; polyamine; stress response.

FIG 1

Salt stress-induced attachment of Synechocystis on glass. (A) Synechocystis wild-type culture. Synechocystis cells were grown in BG11 medium without (upper) or with (bottom) 500 mM NaCl. (B) Effect of NaCl, KCl, or sorbitol on biofilm formation in cells grown in 96-well polystyrene plates. The values shown are the absorbances (OD₅₉₀) of crystal violet extracted from stained biofilms remaining in the wells after removal of the culture. Each column represents the mean ± standard deviation (SD) of data obtained from 77 to 80 individual cultures. Significant differences between no addition and each condition were analyzed by Student's t test (***, P < 0.001). (C) Autofluorescence of Synechocystis cells imaged by CLSM was used as a measure of biofilm formation. Higher autofluorescence was seen in cells grown in medium with added NaCl (250 mM and 500 mM). CLSM images shown are representative images of experiments performed in triplicate. (D) FE-SEM images of cells within the biofilms formed under the same culture conditions as those described for panel C.

FIG 2

Changes in polyamine content in Synechocystis during salt stress. Agmatine, putrescine, spermidine, and spermine contents were determined after addition of NaCl to the medium (white bars, no NaCl added; light gray bars, 250 mM NaCl; dark gray bars, 500 mM NaCl). Cells were grown for 5 days in the medium indicated. Each value corresponds to the average ± SD (n = 3 or 4). Significant differences between no addition and each added NaCl concentration were analyzed by Student's t test (*, P < 0.05; ***, P < 0.001).

FIG 3

Enzymatic activities of Adc1 and Adc2. (A) Putative pathway of polyamine synthesis from arginine in Synechocystis. The enzymes for the biosynthesis of polyamines from arginine in Synechocystis are indicated (13, 14, 23) according to the information available from CyanoBase (http://genome.microbedb.jp/cyanobase). (B) Histidine-tagged Adc1 and Adc2 were expressed in E. coli, affinity purified, separated by SDS-PAGE on a 10% polyacrylamide gel, and stained with Coomassie brilliant blue R-250. (C) The amino acid sequences of tryptic peptides of Adc1 (top) and Adc2 (bottom) isolated from E. coli are highlighted in red. (D) Measurement of arginine decarboxylase activity of Adc1 and Adc2 at various pH values. Each data point corresponds to the average ± SD (n = 3). ND stands for not detected.

FIG 4

Expression and enzymatic activity of Adc1 and Adc2 during salt stress. (A) adc1 and adc2 promoter activities were measured by determining the β-galactosidase activity of the corresponding reporter strain during salt stress (white bars, no NaCl added; gray bars, 500 mM NaCl). Each value corresponds to means ± SD (n = 3). No significant differences (P > 0.05) were found by Student's t test. (B) Semiquantitative RT-PCR to determine adc1 and adc2 expression in Synechocystis during salt stress. (Top) The RT-PCR products of adc1, adc2, and rpnA amplified from total RNA were separated by agarose gel electrophoresis. (Bottom) The band intensity of adc1 and adc2 was determined and related to the band intensity of rpnA to calculate the relative expression level. Each value corresponds to means ± SD (n = 3). No significant differences (P > 0.05) were found by Student's t test. White bars, no NaCl added; gray bars, 500 mM NaCl. (C) Relative arginine decarboxylase activity of Adc1 and Adc2 in the presence of 250 mM (light gray bars) or 500 mM (dark gray bars) NaCl with respect to the activity measured in the buffer without NaCl (white bars). Each data point corresponds to the average ± SD (n = 3).

FIG 5

Spermidine contents in Synechocystis mutants. (A to F) The arrows indicate the positions and orientation of the primers for PCR to confirm the successful disruption or reintroduction of adc1 and adc2 in the Synechocystis genome. The letters on the left correspond to the PCR bands in panels B to F. The overexpression cassette of adc1 (g) or adc2 (h) was integrated at a neutral site, the slr2031 gene. Disruption of the genes in the insertion mutants ko1, kd2, and ko1ko2, obtained by repeated homogeneity segregation, was determined by the presence of a PCR product of the expected length. Note that for adc2 no complete disruption could be obtained (see the text for details). (G) Agmatine and spermidine content of the wild-type (WT) (white bars), ko1 (light gray bars), and ko2 (dark gray bars) strains were measured in cells grown for 5 days in medium without added NaCl. The spermidine content data for the wild type are the same as those shown in Fig. 2. Each value corresponds to the mean ± SD (n = 4). Significant differences between the WT and each mutant were analyzed by Student's t test (*, P < 0.05). No significant differences (P > 0.05) were found in agmatine content by Student's t test. (H) The spermidine contents of ko1ko2, ko1ko2/1ox, and ko1ko2/2ox strains were measured in cells grown for 7 days in medium without added NaCl. Each value corresponds to the mean ± SD (n = 3). Significant differences between ko1ko2 and ko1ko2/1ox or ko1ko2/2ox were analyzed by Student's t test (***, P < 0.001).

FIG 6

Effect of cellular components from Synechocystis on Adc1- or Adc2-mediated arginine decarboxylase activity and spermidine production. (A) Detection of Adc1 and Adc2 expressed in Synechocystis mutants by Western blotting. Arrowheads point to the bands corresponding to histidine-tagged Adc1 (filled arrowhead) and Adc2 (open arrowhead). (B) Total protein isolated from Synechocystis stained with Coomassie brilliant blue R-250. Each lane in panels A and B corresponds to one biological replicate. (C and D) Enzymatic activity measurements. Arginine was added to extracts of ko1ko2, ko1ko2/1ox, and ko1ko2/2ox. Amounts of agmatine (C) and spermidine (D) were determined after 1 h. Each value corresponds to the mean ± SD (n = 3). Significant differences between ko1ko2 and ko1ko2/1ox or ko1ko2/2ox were analyzed by Student's t test (**, P < 0.01). ND stands for not detected.

FIG 7

Effect of inactivation of adc1 and adc2 on growth and biofilm formation in Synechocystis. (A and B) Representative microscopic images obtained by CLSM (A) or FE-SEM (B) show biofilm formation of ko1, kd2, and ko1ko2 strains in medium after 5 days not seen with the wild type. Sample preparation was essentially the same as that described for Fig. 1C and D. The experiments were replicated three times and representative images are shown. (C) Growth curves of Synechocystis wild-type, ko1, kd2, ko1ko2, ko1ko2/1ox, and ko1ko2/2ox strains cultivated in BG11 medium without NaCl addition in 96-well polystyrene plates. (Top) The cell density was measured as the OD₇₃₀. (Bottom) The amount of cells attached to the walls of the plates was quantified by crystal violet staining as described in the legend to Fig. 1. Each value corresponds to the mean ± SD (n = 3). Significant differences between the wild type (WT) and each mutant were analyzed by Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 8

Morphological changes in a Synechocystis culture. During salt stress a change in life style occurs, leading planktonic Synechocystis cells to form cell aggregations in a biofilm. The conversion of arginine to agmatine, which is the initial step in the polyamine synthesis pathway in Synechocystis, is performed by two arginine decarboxylases, Adc1 and Adc2. Spermidine contents decline when the biofilm is formed.