The yydFGHIJ operon of Bacillus subtilis encodes a peptide that induces the LiaRS two-component system

Abstract

The Bacillus subtilis LiaRS two-component system (TCS) responds to perturbations of the cell envelope induced by lipid II-interacting antibiotics, such as vancomycin, ramoplanin, nisin, and bacitracin. Here, we characterize Tn7-generated mutations that induce the liaRS TCS. In addition to insertions in liaF, a known negative regulator of the LiaRS TCS, we identified two disruptions in the last two genes of the yydFGHIJ operon. This operon is predicted to encode a 49-amino-acid peptide (YydF), a modification enzyme (YydG), a membrane-embedded protease (YydH), and an ATP-binding cassette (ABC) transporter (YydIJ). Genome sequence comparisons suggest that the yydFGHIJ operon may have been acquired by horizontal transfer. Inactivation of the YydIJ transporter resulted in increased expression from the LiaR-dependent P(liaI) promoter only in the presence of the yydFGH genes. Cells harboring the complete yydFGHIJ operon induced LiaR activity in cocultured cells lacking either this transporter or the complete operon. These results suggest that this operon is involved in the synthesis and export of a modified peptide (YydF*) that elicits cell envelope stress sensed by the LiaRS TCS.

FIG. 1.

A. Arrangement of the yydFGHIJ operon. The schematic is drawn to scale, and predicted functions are shown above the genes. Large arrows represent the open reading frames, with the P_yydF and P_yydI promoters identified in this study marked. Open triangles show the positions of the Tn7 transposon insertions: 1, YPL3, YPL9, YPL14, and YPL15; 2, YPL6 and YPL13. Also indicated are the positions of the fragments used by Albano et al. for gel shift and pMUTIN construction (1) and the fragments used in this study to create lacZ fusions at the amyE locus. B. The yydF promoter region and yydF-yydG intergenic region. The −35 and −10 regions of a σ^A-type promoter are underlined, and the +1 start of transcription as determined by 5′-RACE PCR is shown in bold. The yydF and yydG open reading frames are indicated by dashes, and the inverted repeat is shown with arrows (with the complementary bases marked in bold).

FIG. 2.

A. Induction of the P_liaI-74-cat-lacZ fusion in WT (black bars), yydIJ::spec (white bars), and yydFGHIJ::spec (gray bars) strains on solid LB, DSM, and MC media. B. Activities of P_sigW, P_sigX, and P_sigM-lacZ fusions in the same strains on MC medium only. After overnight incubation on solid medium, the cells were washed off and β-galactosidase activity was determined. Error bars represent the standard deviations between two independent experiments each assayed in duplicate.

FIG. 3.

Expression from the P_yydF and P_yydI promoters. Expression levels of the P_yydF-lacZ fusion (A and C) and P_yydI-lacZ fusion (B and D) are shown. (A and B) Expression during growth in different media: rich medium (LB; black bars), sporulation medium (DSM; white bars), and MC medium (gray bars). (C and D) Expression from these promoters in the wild type (black bars) and abrB (white bars) and rok (gray bars) mutants grown in LB medium. Note that T₀ corresponds to the transition from logarithmic growth to stationary phase. Each experiment was repeated at least twice, and a representative assay is shown here. Error bars represent standard deviations between triplicate β-galactosidase assays.

FIG. 4.

Northern blot analysis showing that a small (approximately 250-bp) transcript corresponding to the yydF gene is transcribed in liquid LB, MC, and MM media and is increased in stationary phase. The blot was probed with a radiolabeled yydF fragment.

FIG. 5.

Arrangement of yyd operon homologs in other bacteria. Open reading frames are not to scale. Homologs of the yydFGHIJ genes are shaded, while other surrounding genes are shown in white. Bsu, B. subtilis subsp. subtilis strains 168 and RO-NN-1 as well as B. subtilis subsp. spizizenii TU-B-10; Sag, Streptococcus agalactiae (strains indicated; genes encoding putative streptomycin resistance [Strep^r] proteins and NRAMP family Mn²⁺/Fe²⁺ transporters [NRAMP] are also indicated); Bce, B. cereus subsp. cytotoxis NVH 391-98 (the yydH gene is truncated, and the upstream putative integrase [int] and flanking transposases of the IS3 and IS4 families are shown); Sau, Staphylococcus aureus subsp. aureus (predicted hydrolase [hyd] and sortase [sort] genes are also indicated). All sequences can be found at the National Center for Biotechnology website (http://www.ncbi.nlm.nih.gov), except for the unfinished genomes of B. subtilis sp. RO-NN-1 and TU-B-10.