Antibiotic Stimulation of a Bacillus subtilis Migratory Response

Abstract

Competitive interactions between bacteria reveal physiological adaptations that benefit fitness. Bacillus subtilis is a Gram-positive species with several adaptive mechanisms for competition and environmental stress. Biofilm formation, sporulation, and motility are the outcomes of widespread changes in a population of B. subtilis. These changes emerge from complex, regulated pathways for adapting to external stresses, including competition from other species. To identify competition-specific functions, we cultured B. subtilis with multiple species of Streptomyces and observed altered patterns of growth for each organism. In particular, when plated on agar medium near Streptomyces venezuelae, B. subtilis initiates a robust and reproducible mobile response. To investigate the mechanistic basis for the interaction, we determined the type of motility used by B. subtilis and isolated inducing metabolites produced by S. venezuelae. Bacillus subtilis has three defined forms of motility: swimming, swarming, and sliding. Streptomyces venezuelae induced sliding motility specifically in our experiments. The inducing agents produced by S. venezuelae were identified as chloramphenicol and a brominated derivative at subinhibitory concentrations. Upon further characterization of the mobile response, our results demonstrated that subinhibitory concentrations of chloramphenicol, erythromycin, tetracycline, and spectinomycin all activate a sliding motility response by B. subtilis. Our data are consistent with sliding motility initiating under conditions of protein translation stress. This report underscores the importance of hormesis as an early warning system for potential bacterial competitors and antibiotic exposure. IMPORTANCE Antibiotic resistance is a major challenge for the effective treatment of infectious diseases. Identifying adaptive mechanisms that bacteria use to survive low levels of antibiotic stress is important for understanding pathways to antibiotic resistance. Furthermore, little is known about the effects of individual bacterial interactions on multispecies communities. This work demonstrates that subinhibitory amounts of some antibiotics produced by streptomycetes induce active motility in B. subtilis, which may alter species interaction dynamics among species-diverse bacterial communities in natural environments. The use of antibiotics at subinhibitory concentrations results in many changes in bacteria, including changes in biofilm formation, small-colony variants, formation of persisters, and motility. Identifying the mechanistic bases of these adaptations is crucial for understanding how bacterial communities are impacted by antibiotics.

Keywords: Bacillus subtilis; Streptomyces venezuelae; antibiotics; chloramphenicol; competition; hormesis; ribosome; sliding motility.

FIG 1

S. venezuelae induces B. subtilis mobilization. (A) Different species of Streptomyces were cultured with B. subtilis to identify patterns of interaction. Streptomyces species were spotted in the horizontal line, and B. subtilis was in the vertical line. Pictures were taken at h 40. (B) S. venezuelae (horizontal spots) induced proximal B. subtilis (vertical spots) to migrate across the agar surface, while this migration was not observed in the distal spots. The right panel presents an enlarged view, highlighting the mobile region inside the dashed box. The picture was taken at h 18. Bars, 1 cm.

FIG 2

Identification of S. venezuelae-induced mobility as sliding. S. venezuelae was spotted in the horizontal line in both panels A and B. Pictures were taken at h 48. (A) The mobilization induced by S. venezuelae was observed at up to 2% agar. (B) Different B. subtilis mutants were cultured with S. venezuelae. The mobility of hag mutant was induced but was not observed in either epsH mutants or srfAA mutants. However, when epsH and srfAA were mixed, the mixture was able to mobilize upon challenge with S. venezuelae. Pictures were taken at h 24. Bars, 1 cm.

FIG 3

Identification of monobromamphenicol as a sliding inducer. (A) Crude extract from S. venezuelae agar plates was loaded into the wells near B. subtilis and induced robust sliding motility compared with the medium-only control. All time-based HPLC fractions were collected and tested for activity. One fraction had the sliding inducing activity, and one fraction had the growth inhibitory activity. Pictures were taken at h 24. (B) The inducing fraction was brominated chloramphenicol (X = Br [monobromamphenicol]). The inhibitory fraction was chloramphenicol (X = Cl). Bar, 1 cm.

FIG 4

Chloramphenicol induced B. subtilis sliding at subinhibitory concentrations. (A) The chloramphenicol fraction was 2-fold serially diluted, and 10 μl of each dilution was applied onto a filter paper disc 0.6 cm away from B. subtilis. The control was the 40% (vol/vol) methanol solvent. (B) Pure chloramphenicol was serially diluted and added to the agar plate. At 1 µM, the maximal sliding response was induced. The control was the plate without chloramphenicol. Pictures were taken at h 24. Filter disc diameter, 0.6 cm. Bar, 1 cm.

FIG 5

The ribosome plays a key role in antibiotic-induced sliding. (A) Wild-type strain NCIB 3610 and chloramphenicol (Cm)-resistant strain Cm^r were spotted on the agar plate in the absence (-) or presence (+) of Cm (0.3 µg/ml). (B) Wild-type strain NCIB 3610 and erythromycin (Erm)-resistant strain Erm^r were spotted on the agar plate in the absence or presence of Erm (10 µl of 12.5 µg/ml solution). Pictures were taken at h 24. Filter disc diameter, 0.6 cm. Bar, 1 cm.

FIG 6

bmrCD is related to translation stress but is not required for sliding. (A) Quantitative RT-PCR of bmrCD transcript of the wild-type (WT) strain in the absence (-) and presence (+) of chloramphenicol (Cm) at the indicated time points, 4 h, 6 h, 12 h, and 24 h. Quantification cycle (C_q) values were normalized to C_q values for gyrB. Fold expression values are reported relative to the value for the 4-h sample in the absence of Cm. (B) The WT NCIB 3610 strain and a bmrCD deletion strain were spotted on the agar plate in the absence or presence of Cm (0.3 µg/ml). Pictures were taken at h 24. Bar, 1 cm.

FIG 7

Summary model for concentration-dependent effects of chloramphenicol on B. subtilis. The competitive culture format for S. venezuelae and B. subtilis suggests a model for the spatial and temporal effects of population growth on production and diffusion of chloramphenicol in the agar medium. (A) Early (~24 h) development of the S. venezuelae strain (light green spots) results in low concentrations (yellow) of chloramphenicol in the medium, sufficient for stimulating sliding motility in the proximal B. subtilis strain (light tan shapes). (B) Continued growth (~48 h) and, presumably, chloramphenicol biosynthesis by the proximal S. venezuelae spot are impeded by the migratory population of B. subtilis. During this time, the more distal spots of S. venezuelae grow to a greater extent and produce higher yields of chloramphenicol. The concentration of chloramphenicol (and possibly other, unidentified metabolites) becomes sufficient (red) to impede growth and progression of the sliding population of B. subtilis, which is therefore prevented from contacting the S. venezuelae population. The unaffected populations of B. subtilis (not mobilized by chloramphenicol exposure) are visible as dark tan spots.