Studies of the effect of indole on bacterial cells have been focused on E. coli, which produces indole. High temperature (50 °C), low pH, and the presence of the antibiotics affect indole production in E. coli (Han et al., 2011). Thus, indole production may be affected by the surrounding environment, and this will in turn affect bacterial communities and their life. The impact of exogenous indole on the physiological characteristics of non-indole-producing bacteria has been poorly explored. Indole production during mixed-culture growth between P. aeruginosa and E. coli prevented pyocyanin production and quorum sensing–regulated virulence factors in P. aeruginosa (Chu et al., 2012). Indole could be degraded by various oxygenases from bacteria, fungi, and plants (Lee & Lee, 2010) and quickly reduced in P. aeruginosa (Lee et al., 2009). Degradation and incorporation of indole into tryptophan biosynthesis may be the first mechanism to prevent stress caused by indole. Our microarray data also showed the highest expression of trpB genes in the presence of indole (Table S2). If non-indole-producing bacteria cannot degrade or use excess indole, defense mechanisms such as molecular chaperones and proteases are turned on. Our data demonstrate that indole increases biofilm formation and reduces cell size in P. putida. These results are inconsistent with previously well-known studies using E. coli strains. We speculate that the roles of indole may differ between Pseudomonas species and indole-producing bacteria. Among indole derivatives, indole-3-acetic acid (IAA) has been extensively studied in terms of a symbiotic relationship with soil bacteria and plants. When E. coli K-12 was treated with IAA, the majority of genes encoding cell envelope components and adaptation-related proteins were differentially expressed (Bianco et al., 2006a, b). IAA-treated cells had enhanced biofilm formation due to an increased production of lipopolysaccharide (LPS) and exopolysaccharide. In addition, IAA induced higher levels of the heat-shock protein DnaK, as also seen in our data. In E. coli, sigma factor, σ32, encoded by the rpoH gene, positively regulates the induction of heat-shock proteins such as DnaK, DnaJ and GrpE, and GroEL/ES (Kobayashi et al., 2011). These heat-shock proteins have been extensively studied in other bacterial cells. Many heat-shock proteins, which were highly expressed at high temperatures, include hslU, hslV, htpG, grpE, dnaK, ibpA, clpB, lon, and hflK in E. coli (Richmond et al., 1999); htpG, grpE, dnaK, groEL, and groES in Bacillus subtilis (Helmann et al., 2001); dnaK, groEL, groES, clpB, and lon in Mycoplasma pneumonia (Weiner et al., 2003); and ibpA, groEL, and groES in Yersinia pestis (Motin et al., 2004). Many of these heat-shock proteins were highly expressed in our microarray analysis with indole (Table. S2) and are reported to be expressed under various environmental stresses, such as nutrient starvation, exposure to pollutants, and changes in pH or osmolarity (Koide et al., 2006). In case of P. putida KT2440, toluene or o-xylene treatment induced those genes, such as groES, groEL, lon-1, lon-2, ibpA, htpG, danK, grpE, hslV, and hslU (Domínguez-Cuevas et al., 2006).
Although indole has been reported to cause oxidative stress (Vega et al., 2012), our microarray data did not show any change in oxidative stress–related genes. To investigate the degree of cellular oxidative stress in the presence of indole, superoxide production was measured using the nitroblue tetrazolium (NBT) assay. Exponentially growing cells were treated with different concentrations of indole and ethanol for 30 min. Concentrations of 1–3 mM indole did not cause oxidative stress (data not shown). Therefore, indole probably stresses bacterial cells by disrupting the electron transport system or energy generation and protein folding, rather than by producing oxidative stress, as seen in our study.
In this study, we have shown that indole, at concentrations above a certain level that appears to be toxic to non-indole-producing Pseudomonas strains, altered the expression of many genes in three functional categories: (1) proteases; (2) molecular chaperones; and (3) TCA cycle enzymes. These gene products play important roles in conditions of indole-induced stress. The expression of these genes might cause phenotypic and physiological changes such as in cell morphology, biofilm formation, the NADH/NAD+ ratio, and ATP concentrations. Our data provide evidence that indole, which has been recently spotlighted as a beneficial signal molecule for E. coli, also exerts deleterious effects on non-indole-producing Pseudomonas strains.