Three types of taxis used in the response of Acidovorax sp. strain JS42 to 2-nitrotoluene

Abstract

Acidovorax sp. strain JS42 is able to utilize 2-nitrotoluene (2NT) as its sole carbon, nitrogen, and energy source. We report here that strain JS42 is chemotactic to 2NT and that the response is increased when cells are grown on compounds such as 2NT that are known to induce the first step of 2NT degradation. Assays with JS42 mutants unable to oxidize 2NT showed that the first step of 2NT metabolism was required for the induced response, but not for a portion of the constitutive response, indicating that 2NT itself is an attractant. The 2NT metabolite nitrite was shown to be a strong attractant for strain JS42, and sufficient nitrite was produced during the taxis assay to account for a large part of the induced response. A mutant with an inactivated ntdY gene, which is located adjacent to the 2NT degradation genes and codes for a putative methyl-accepting chemotaxis protein, showed a defect in taxis toward 2NT that may involve a reduced response to nitrite. Responses of a mutant defective for the energy-taxis receptor, Aer, indicated that a functional aer gene is required for a substantial part of the wild-type induced response to 2NT. In summary, strain JS42 utilizes three types of taxis to sense and respond to 2NT: constitutive 2NT-specific chemotaxis to directly sense 2NT, metabolism-dependent nitrite-specific chemotaxis that may be mediated by NtdY, and energy taxis mediated by Aer.

Fig 1

2-Nitrotoluene (2NT) degradation pathway in Acidovorax sp. strain JS42. 2NTDO, 2-nitrotoluene 2,3-dioxygenase, encoded by ntdAaAbAcAd. The catechol degradation (Ctd) enzymes were identified based on the analysis of the JS42 genome sequence (23). CtdE1, catechol 2,3-dioxygenase; CtdF, 2-hydroxymuconate semialdehyde hydrolase; CtdJ, 2-oxopent-4-dienoate hydratase; CtdK, 4-hydroxy-2-oxovalerate aldolase; CtdQ, acetaldehyde dehydrogenase (acylating); 3MC, 3-methylcatechol; HOD, 2-hydroxy-6-oxohepta-2,4-dienoate; HPD, 2-hydroxypenta-2,4-dienoate; HO, 4-hydroxy-2-oxovalerate; AA, acetaldehyde.

Fig 2

Wild-type JS42m responses in the qualitative capillary assay. Responses to buffer (negative control) or succinate (10 mM; positive control) (A) or to 2NT (saturated; ∼4.4 mM) after growth with succinate alone (10 mM) compared to growth in the presence of possible inducers (B) are shown. The concentration of all inducer compounds was 500 μM. 3MC, 3-methylcatechol; 2NBA, 2-nitrobenzyl alcohol. The responses of all cultures to 10 mM succinate were comparable (data not shown).

Fig 3

Chemotactic response to nitrite and generation of nitrite from 2NT during the qualitative capillary assay performed with wild-type JS42m. (A) Responses to ∼4.4 mM nitrite by uninduced and 2NT-induced JS42m. (B) Determination of the amounts of nitrite accumulating at the tips of capillaries containing various nitrite concentrations after 20 min. Approximately 10 μM nitrite accumulated at the tip of a capillary containing 2.5 mM nitrite (arrows). The presence or absence of cells did not affect the accumulation of nitrite. The limit of detection of the nitrite assay is approximately 2 μM. (C) Comparison of induced responses of JS42m to 2NT (∼4.4 mM) and 2.5 mM nitrite. The 2.5 mM nitrite in the capillary corresponds to the approximate amount of nitrite generated by induced cells from 4.4 mM 2NT during a 20-min assay. (D) Comparison of uninduced responses of JS42m to 2NT (∼4.4 mM) and 0.5 mM nitrite. The 0.5 mM nitrite in the capillary corresponds to approximately 4 μM nitrite in solution at the mouth of the capillary (see panel B).

Fig 4

Chemotactic response to 2NT (∼4.4 mM) in the qualitative capillary assay by JS42m and mutant derivatives following growth in uninduced and 2NT-induced conditions. Photographs were taken after 20 min. The response after 60 min is also shown for strain CAR22, since the peak response was delayed. All strains showed a strong response to succinate (positive control; data not shown).

Fig 5

Tactic responses to 2NT (∼4.4 mM) of the wild type (JS42m), ntdY mutant (CAR3), complemented ntdY mutant (CAR3[pCAR27]), ntdAc mutant (CAR20), and ntdAc mutant expressing the nbzY gene (CAR20[pCAR27]) in the qualitative capillary assay after growth in uninduced and 2NT-induced conditions and to the 2NT metabolite nitrite (4.4 mM) after growth in uninduced conditions. The responses of all strains to 10 mM succinate were comparable (data not shown).

Fig 6

Responses of the wild type (JS42m), aer deletion mutant (CAR5), and complemented aer mutant (CAR5[pCAR9]) in soft agar plates containing 2 mM 2NT after incubation for 2 days at 30°C. Strain CAR24 (cheA::Km) was used as a negative control. (A) Representative 2NT soft agar plate. (B) Averaged colony diameters in 2NT soft agar plates (n = 6; error bars represent standard deviations). Different letters indicate significantly different means as calculated by one-way analysis of variance (ANOVA) (Tukey multiple comparison test; P < 0.05).

Fig 7

Responses of the wild type and the receptor mutants to 2NT. (A) Qualitative capillary assay results (∼4.4 mM 2NT) after growth under uninduced and 2NT-induced conditions. (B) Representative soft agar plate containing 2 mM 2NT after incubation for 3 days at 30°C. Strain CAR24 (cheA::Km) was used as a negative control. (C) Averaged colony diameters in 2NT soft agar plates (n = 4; error bars represent standard deviations). Different letters indicate significantly different means as calculated by one-way ANOVA (Tukey multiple comparison test; P < 0.05).