Activity of the osmotically regulated yqiHIK promoter from Bacillus subtilis is controlled at a distance

Abstract

The yqiHIK gene cluster from Bacillus subtilis is predicted to encode an extracellular lipoprotein (YqiH), a secreted N-acetylmuramoyl-L-alanine amidase (YqiI), and a cytoplasmic glycerophosphodiester phosphodiesterase (YqiK). Reverse transcriptase PCR (RT-PCR) analysis showed that the yqiHIK genes are transcribed as an operon. Consistent with the in silico prediction, we found that the purified YqiI protein exhibited hydrolytic activity toward peptidoglycan sacculi. Transcription studies with yqiH-treA reporter fusion strains revealed that the expression of yqiHIK is subjected to finely tuned osmotic control, but enhanced expression occurs only in severely osmotically stressed cells. Primer extension analysis pinpointed the osmotically responsive yqiHIK promoter, and site-directed mutagenesis was employed to assess functionally important sequences required for promoter activity and osmotic control. Promoter variants with constitutive activity were isolated. A deletion analysis of the yqiHIK regulatory region showed that a 53-bp AT-rich DNA segment positioned 180 bp upstream of the -35 sequence is critical for the activity and osmotic regulation of the yqiHIK promoter. Hence, the expression of yqiHIK is subjected to genetic control at a distance. Upon the onset of growth of cells of the B. subtilis wild-type strain in high-salinity medium (1.2 M NaCl), we observed gross morphological deformations of cells that were then reversed to a rod-shaped morphology again when the cells had adjusted to the high-salinity environment. The products of the yqiHIK gene cluster were not critical for reestablishing rod-shaped morphology, but the deletion of this operon yielded a B. subtilis mutant impaired in growth in a defined minimal medium and at high salinity.

Fig 1

Structure and genetic organization of the yqiHIK gene cluster. (A) Genetic map of the yqiHIK genes in the genome of B. subtilis. The length and the position of the different PCR-amplified fragments are indicated. A bent arrow indicates the promoter region and the direction of yqiHIK transcription; the position of a putative factor-independent transcriptional terminator sequence is marked by a lollipop. (B) RT-PCR-based analysis of the putative yqiHIK operon. The amplification reactions using the indicated DNA primers (P1 to P4) were conducted on different templates: cDNA, genomic DNA as a positive control, and RNA as a negative control. Total RNA was isolated from cells of B. subtilis strain 168 grown in SMM. M, molecular marker.

Fig 2

Cell wall hydrolase activity of the purified YqiI–Strep-tag-II protein. (Left) Coomassie blue-stained SDS-PAGE gel of the affinity purified YqiI–Strep-tag-II protein. (Right) Zymogram analysis (16) using purified B. subtilis 168 sacculi. Staining of the polyacrylamide gel with methylene blue and destaining with distilled water detected areas of peptidoglycan lysis. Eight micrograms of the purified YqiI–Strep-tag-II protein was applied for both SDS-PAGE and the zymogram.

Fig 3

In silico analysis of the YqiI protein. (A) Alignment of the amino acid (AA) sequences (catalytic domain only) of the YqiI, LytC, CwlC, CwlD, and YrvJ proteins from B. subtilis and the CwlV1 protein from P. polymyxa var. colistinus. Black boxes highlight amino acids that are involved in the coordination of a zinc ion in the active site of amidases (15, 54). (B) Domain organization of B. subtilis proteins homologous to YqiI. The lengths of the proteins are indicated. Boxes with hatch marks represent Sec-type signal sequences; they are present in all amidases shown except for CwlC. The amidase_3 (Ami_3) domain (PF015020) is marked. Black boxes and gray triangles represent different types of cell wall-binding domains: cell wall-binding domain 2 (CWB_2) (PF04122) of LytC, the spore domain (SPOR) of CwlC (PF05036), the SH3b domain of YrvJ (SM00287), and the amine domain (AMIN) (PF11741) of CwlV. (C) In silico model of YqiI based on the crystal structure of the catalytic domain of the CwlV1 protein (PDB accession number 1JWQ) from P. polymyxa var. colistinus. The amino acids H-15, E-29, H-82, and E-148 are predicted to coordinate a zinc ion in the active center of the protein and contribute to enzyme activity.

Fig 4

Growth properties of yqiHIK deletion mutant strain KFB8 and strain KFB37 (lytC cwlC cwlD yrvJ yqiI). (A and B) Cultures of B. subtilis wild-type strain 168 (black circles), its Δ(yqiHIK::neo) derivative strain KFB8 (white circles), and the lytC cwlC cwlD yrvJ yqiI quintuple mutant strain KFB37 (gray circles) were grown in SMM alone (A) or in SMM containing 1.2 M NaCl (B). (C) The morphologies of B. subtilis strains 168, KFB8, and KFB37 were analyzed by phase-contrast microscopy at different time points (indicated by arrows) during the growth of the cultures. Scale bar, 5 μm.

Fig 5

Fluorescence and scanning electron microscopy of salt-stressed B. subtilis cells. (A) Cells of wild-type strain 168 and its mutant derivatives KFB8 (yqiHIK) and KFB37 (lytC cwlC cwlD yrvJ yqiI) were grown in SMM with 1.2 M NaCl for 12 h (time point 4) (Fig. 4B) and observed by both phase-contrast and fluorescence (after staining with the Live/Dead BacLight bacterial viability kit) microscopy. Scale bar, 5 μm. (B and C) Cultures of strain 168 were grown to the early exponential phase (OD₅₇₈ of 0.7 to 0.9) in SMM alone (B) or in SMM containing 1.2 M NaCl (C) and were observed by scanning electron microscopy. The magnification was set at ×8,000, and the scale bar represents 4 μm.

Fig 6

Influence of the AT-rich region located upstream of the yqiHIK promoter region on osmotic induction of yqiH-treA reporter gene expression. (A) DNA sequence of the 300-bp fragment fused to the promoterless treA reporter gene in strain KFB15. The AT-rich region is highlighted by a black box. The DNA fragment shown is defined as Δ0. Arrows indicate three different truncations (Δ1 to Δ3) of the 300-bp region originally used to construct the yqiH-treA gene fusion. The positions of the −35 and −10 yqiHIK promoter regions, the transcriptional initiation site, the ribosome-binding site of the yqiH gene, and its translational start codon of the yqiH reading frame are indicated. (B) Promoter activity and osmoregulation of reporter strains carrying the various deletion constructs in cells cultivated either in SMM or in SMM containing 1.2 M NaCl.

Fig 7

Osmotic regulation of yqiH-treA expression and identification of the yqiHIK transcription start site by primer extension analysis. (A) Determination of TreA activity in cells of strain KFB15 (yqiH-treA) cultured in SMM with increasing NaCl concentrations. All cell samples were harvested for TreA enzyme activity measurements at the mid-exponential growth phase (OD₅₇₈ of about 1.0 to 1.5). (B) Primer extension analysis of the yqiHIK transcript in cells of B. subtilis strain 168 carrying plasmid pKF16 (yqiH′) cultured either in SMM (−) or in SMM with 1.2 M NaCl (+). (C) Nucleotide sequence of the yqiHIK promoter region. The transcription initiation site (+1) is indicated by a bent arrow. The −35 and −10 regions are highlighted with gray boxes and are separated by 17 bp. The ribosome-binding site (RBS) of the yqiH gene, its start codon, and the N-terminal amino acid sequence of the YqiH protein are shown.