Gene yerP, involved in surfactin self-resistance in Bacillus subtilis

Abstract

Surfactin is a cyclic lipopeptide biosurfactant. Transposon mutagenesis was performed in Bacillus subtilis strain 168, and a surfactin-susceptible mutant, strain 801, was isolated. Analysis of the region of insertion revealed that yerP was the determinant of surfactin self-resistance. YerP had homology with the resistance, nodulation, and cell division (RND) family proton motive force-dependent efflux pumps only characterized in gram-negative strains. The yerP-deficient strain 802, in which the internal region of the yerP gene of B. subtilis strain 168 was deleted, showed susceptibility to acriflavine and ethidium bromide. When strain 802 was converted to a surfactin producer by introducing a functional sfp which encodes a 4'-phosphopantetheinyl transferase and is mutated in B. subtilis strain 168, this yerP-deficient strain produced surfactin, although surfactin production was significantly reduced. The expression of yerP was at its maximum at the end of the logarithmic growth phase and was not induced by surfactin. yerP is the first RND-like gene characterized in gram-positive strains and is supposed to be involved in the efflux of surfactin.

FIG. 1

(A) Physical and genetic map of the yerP-yerO region of yerP-deficient strains derived from B. subtilis strain 168. The locations of the yerO and yerP coding regions (Z99107) are indicated by arrows pointing in the direction of transcription. The positions of the relevant restriction sites are indicated. (B) Photographs of growth inhibition of 6-h cultures of strains 801, 802, and 168 on L-agar-surfactin plates with various surfactin concentrations after a 12-h incubation in an incubator at 37°C.

FIG. 2

(A) Determination of the transcriptional start site of yerP by primer extension. The arrow indicates the band corresponding to the yerP-specific primer extension product. (B) The nucleotide sequence of the intergenetic yerO-yerP region, the nontranscribed yerO strand and the transcribed yerP strand, is shown in the 5′-to-3′ direction. The deduced primary structure of the polypeptide encoded by yerP is shown in the single-letter code below the nucleotide sequence. The yerP transcriptional start site (+1) defined by the primer extension method, the −35 and −10 regions of the promoter, and the putative ribosome binding site are indicated below the nucleotide sequence. > < indicates an inverted repeat sequence. The complementary sequence of the primer, used in the primer extension method, is underlined.

FIG. 3

Drug and surfactin susceptibility of yerP-deficient strains 801 and 802. Bacteria were grown in L medium for 6 or 24 h. Four microliters of 10⁰ through 10⁵ serial dilutions were spotted on L-agar-drug and L-agar-surfactin plates and incubated at 37°C for 12 h. The initial numbers of bacteria were as follows: strain 168 (6 h), 7.0 × 10⁸ CFU/ml; strain 801 (6 h), 6.1 × 10⁸ CFU/ml; strain 802 (6 h), 5.4 × 10⁸ CFU/ml; strain 168 (24 h), 2.4 × 10⁸ CFU/ml; strain 801 (24 h), 2.0 × 10⁸ CFU/ml; and strain 802 (24 h), 3.7 × 10⁸ CFU/ml. In the 10⁰ spot (nondilution spot) of all strains, no colony was observed at concentrations higher than following: acriflavine, 5 μg/ml; ethidium bromide, 10 μg/ml; tetracycline, 10 μg/ml; Triton X-100, 500 μg/ml; and SDS, 250 μg/ml. Only one result for each drug is shown.

FIG. 4

Surfactin susceptibility of strains 801 (▵), 802 (□), and 168 (○). The 6-h (A) and 24-h (B) cultures were plated on L agar-surfactin plates containing different surfactin concentrations, and the survival rates were measured after 24 h of incubation. Error bars represent standard deviations of the means (n = 3).

FIG. 5

Growth (A and B) and expression (C and D) of yerP-lacZ and yerO-lacZ in L medium alone (A and C) or supplemented with surfactin (500 μg/ml) (B and D). The change in expression of yerP-lacZ and yerO-lacZ was determined by β-galactosidase specific activity and calculated in Miller units and is shown plotted against the OD₆₀₀. ○, strain 803 (yerP⁺/yerP-lacZ); □, strain 804 (yerP⁺/yerO-lacZ); ●, strain 806 (ΔyerP/yerP-lacZ); ▪, strain 807 (ΔyerP/yerO-lacZ). The expression levels of the control strains inserted promoterless lacZ into the amyE gene of both the yerP⁺ (strain 805) and ΔyerP (strain 808) backgrounds were lower than 2 Miller units at each measured point (data not shown).