Lon protease of Azorhizobium caulinodans ORS571 is required for suppression of reb gene expression

Abstract

Bacterial Lon proteases play important roles in a variety of biological processes in addition to housekeeping functions. In this study, we focused on the Lon protease of Azorhizobium caulinodans, which can fix nitrogen both during free-living growth and in stem nodules of the legume Sesbania rostrata. The nitrogen fixation activity of an A. caulinodans lon mutant in the free-living state was not significantly different from that of the wild-type strain. However, the stem nodules formed by the lon mutant showed little or no nitrogen fixation activity. By microscopic analyses, two kinds of host cells were observed in the stem nodules formed by the lon mutant. One type has shrunken host cells containing a high density of bacteria, and the other type has oval or elongated host cells containing a low density or no bacteria. This phenotype is similar to a praR mutant highly expressing the reb genes. Quantitative reverse transcription-PCR analyses revealed that reb genes were also highly expressed in the lon mutant. Furthermore, a lon reb double mutant formed stem nodules showing higher nitrogen fixation activity than the lon mutant, and shrunken host cells were not observed in these stem nodules. These results suggest that Lon protease is required to suppress the expression of the reb genes and that high expression of reb genes in part causes aberrance in the A. caulinodans-S. rostrata symbiosis. In addition to the suppression of reb genes, it was found that Lon protease was involved in the regulation of exopolysaccharide production and autoagglutination of bacterial cells.

Fig 1

Autoagglutination activity of the lon mutant and the lon reb double mutant. ORS571 (wild type [WT]), Anx171 (Δlon), Anx178 (Δlon Δreb), and Anx7 (ΔpraR) were grown in L3+N medium under aerobic conditions in vertical test tubes in a rotary shaker. After 16 h of incubation, cultures were observed.

Fig 2

EPS production of the lon mutant. (A) Colonies of ORS571 (wild type [WT]) and Anx171 (Δlon) grown for 3 days on TY or L3+N medium plates containing 0.8% agar. A 15-μl aliquot of the bacterial suspension (OD₆₀₀ of 1.0) was spotted onto each plate. (B) Measurement of EPS produced by ORS571 (wild type [WT]) and Anx171 (Δlon). The colonies on the plates were collected, and the EPSs released from bacterial cells were quantified. The EPS amount from each colony was evaluated by normalizing the collected cell suspension to the OD₆₀₀. The values are means ± standard deviations of five replicate colonies. Different letters indicate significant differences (P < 0.05; Tukey-Kramer).

Fig 3

Stem nodules formed by the lon mutant and the lon reb double mutant. (A) Inner regions of stem nodules formed by ORS571 (wild type [WT]), Anx171 (Δlon), Anx178 (Δlon Δreb), and Anx7 (ΔpraR). Each strain was inoculated onto the stems of S. rostrata plants, and the stem nodules were observed at 7 and 12 dpi. (B) Nitrogen fixation activities of the stem nodules. The stem nodules formed by each strain were harvested at 7 to 14 dpi, and ARA was measured. The values are means ± standard deviations of five replicate plants. Different letters above the bars indicate significant differences (P < 0.05; Tukey-Kramer).

Fig 4

Optical microscope images of stem nodules of ORS571, Anx171 (Δlon), Anx7 (ΔpraR), and Anx78 (Δlon Δreb). Stem nodules at 7 dpi (A to H) and 12 dpi (I to O) were longitudinally sectioned and stained with toluidine blue O. Panels: A, E, and I, ORS571; B, F, J, and M, Anx171; C, G, K, and N, Anx7; D, H, L, and O, Anx178. Abbreviations: oc, oval or elongated host cell; sc, shrunken host cell; uc, unstained host cell; v, vacuole.

Fig 5

Quantitative RT-PCR analyses of reb genes, exp genes, and praR in the free-living and symbiotic states. (A) Expression of reb genes (AZC_3781, AZC_3782, AZC_3783, and AZC_3786). (B) Expression of exp genes (AZC_3319, AZC_3325, AZC_3326, AZC_3328, AZC_3329, and AZC_3331). (C) Expression of praR (AZC_0013). Total RNAs were isolated from the free-living bacteria of strains ORS571 (wild type [WT]) and Anx171 (Δlon) grown in TY and L3+N media under aerobic conditions and from the stem nodules formed by ORS571 and Anx171 at 14 dpi. The amounts of transcripts of each gene and 16S rRNA in the total RNA from each sample were estimated by quantitative RT-PCR, and the expression levels of each gene were evaluated by normalization to the 16S rRNA level. The values are means ± standard deviations of three replicate cultures or plants and are presented relative to the results of free-living ORS571 bacteria grown in TY medium.