Specific targeting of the metallophosphoesterase YkuE to the bacillus cell wall requires the twin-arginine translocation system

Abstract

The twin-arginine translocation (Tat) pathway is dedicated to the transport of fully folded proteins across the cytoplasmic membranes of many bacteria and the chloroplast thylakoidal membrane. Accordingly, Tat-dependently translocated proteins are known to be delivered to the periplasm of Gram-negative bacteria, the growth medium of Gram-positive bacteria, and the thylakoid lumen. Here, we present the first example of a protein, YkuE of Bacillus subtilis, that is specifically targeted by the Tat pathway to the cell wall of a Gram-positive bacterium. The cell wall binding of YkuE is facilitated by electrostatic interactions. Interestingly, under particular conditions, YkuE can also be targeted to the cell wall in a Tat-independent manner. The biological function of YkuE was so far unknown. Our present studies show that YkuE is a metal-dependent phosphoesterase that preferentially binds manganese and zinc.

FIGURE 1.

Subcellular localization of YkuE. To determine the subcellular localization of YkuE in the presence or absence of active Tat translocases, the parental strain B. subtilis 168 and the tat mutant strains tatAyCy, tatAdCd, total-tat3, tatAyCy pCACy, and total-tat3 pCACy were transformed with plasmid pHBykuEmyc for the expression of YkuE. Next, the cells producing YkuE were grown for 7 h in LB medium. The cells were then separated from the growth medium by centrifugation and subjected to subcellular fractionation. Proteins in the obtained fractions were separated by PAGE, and the presence of YkuE and YfkN was monitored by Western blotting with specific polyclonal antibodies. Protein loading on the gels was corrected for A₆₀₀. Only the results for the cytosol, membrane, and cell wall fractions are shown because no YkuE was detectable in the fractions representing the growth medium. The cell wall-localized protein YfkN was used as a Tat-independent control. The lanes are labeled as follows: 168, B. subtilis 168 Marburg strain; ykuE, B. subtilis 168 with a disrupted ykuE gene; 168 pHBykuEmyc, B. subtilis 168 containing pHBykuEmyc; AyCy pHBykuEmyc, B. subtilis tatAyCy containing pHBykuEmyc; AdCd pHBykuEmyc, B. subtilis tatAdCd pHBykuEmyc; Total-tat pHBykuEmyc, B. subtilis lacking all tat genes but containing pHBykuEmyc; AyCy pHBykuEmyc pCACy, B. subtilis tatAyCy containing pHBykuEmyc and plasmid pCACy for expression of TatAyCy; Total-tat pHBykuEmyc pCACy, B. subtilis lacking all chromosomal tat genes but containing pHBykuEmyc and pCACy. The positions of mature YkuE (YkuE) and the precursor and mature forms of YfkN (pre-Yfkn, YfkN) are marked with arrows. Asterisks mark bands that aspecifically cross-react with anti-YKuE.

FIGURE 2.

Subcellular localization of YwbN. To determine the subcellular localization of YwbN in the presence or absence of active Tat translocases, the parental strain B. subtilis 168 and tat mutant strains were subjected to subcellular fractionation as described in the legend of Fig. 1. The presence of YwbN, the Tat-independently cell wall-localized control protein YfkN, and the Tat-independently secreted control protein FeuA was monitored by Western blotting with specific polyclonal antibodies. The lanes are labeled as follows: ywbN, B. subtilis 168 with a disrupted ywbN gene; 168, B. subtilis 168 Marburg strain; AyCy, B. subtilis tatAyCy; AdCd, B. subtilis tatAdCd; Total-tat, B. subtilis lacking all tat genes; AyCy pCACy, B. subtilis tatAyCy containing plasmid pCACy for expression of TatAyCy; Total-tat pCACy, B. subtilis lacking all chromosomal tat genes but containing pCACy. The positions of mature YwbN (YwbN), the precursor, and mature forms of YfkN (pre-Yfkn and YfkN) and secreted FeuA (FeuA) are marked with arrows.

FIGURE 3.

Partially Tat-independent cell wall localization of YkuE under phosphate starvation conditions. To investigate the effects of phosphate starvation on the localization of YkuE, cells containing pHBykuEmyc for the production of YkuE were grown in low phosphate depletion media and subjected to subcellular fractionation as described for Fig. 1. The presence of YkuE, YfkN, TatAy, and TrxA was monitored by Western blotting with specific polyclonal antibodies. The lanes are labeled as follows: 168, B. subtilis 168; 168 + tatAy, B. subtilis 168 containing pCAy for expression of tatAy; 168 + tatCy, B. subtilis 168 containing pCCy for expression of tatCy; 168 + tatAyCy, B. subtilis 168 containing pCACy for expression of tatAyCy; AyCy, B. subtilis tatAyCy; AyCy + tatAy, B. subtilis tatAyCy containing pCAy for expression of tatAy; AyCy + tatCy, B. subtilis tatAyCy containing pCCy for expression of tatCy; AyCy + tatAyCy, B. subtilis tatAyCy containing pCACy for expression of tatAyCy; Total-tat, B. subtilis lacking all tat genes; Total-tat + tatAy, B. subtilis lacking all tat genes containing pCAy for expression of tatAy; Total-tat + tatCy, B. subtilis lacking all tat genes containing pCCy for expression of tatCy; Total-tat + tatAyCy, B. subtilis lacking all tat genes containing pCACy for expression of tatAyCy. The positions of mature YkuE (YkuE), a processed form of YfkN (YfkN), TatAy, and TrxA are marked with arrows.

FIGURE 4.

Tat-independent export of overproduced YkuE to the cell wall. To overproduce YkuE, a subtilin-inducible copy of the ykuE gene was expressed from the plasmid pNZ8910ykuE-strep in B. subtilis cells expressing the SpaRK two-component regulatory system. YkuE-overproducing cells grown in LB medium were subjected to subcellular fractionation as described for Fig. 1. The presence of YkuE was monitored by Western blotting with specific polyclonal antibodies. The lanes with samples from YkuE (over)producing cells are labeled as follows: 168, B. subtilis 168; AyCy, B. subtilis tatAyCy; AdCd, B. subtilis tatAdCd; total-tat, B. subtilis lacking all tat genes. YkuE-specific protein bands are marked with an arrow.

FIGURE 5.

LiCl extraction of YkuE from the cell wall. Cells of B. subtilis 168, the ykuE mutant BFA1834, B. subtilis 168 pHBykuE, or B. subtilis SURE ykuE were grown and harvested as described in the legend of Fig. 1. Next, the bacterial cells were treated either with 1.5 m LiCl as described by Antelmann et al. (36) or with lysozyme as described under “Experimental Procedures.” The presence of YkuE or the Tat-independent noncovalently cell wall-bound control protein WprA in the supernatant of LiCl-treated cells or in the protoplast supernatant was monitored by Western blotting with specific polyclonal antibodies. The presence of mature YkuE (YkuE), different processed forms of WprA, and an unidentified protein (X) that cross-reacts with the WprA antibodies are marked with arrows.

FIGURE 6.

YkuE purification and stability in the presence of different protease and hydrolase inhibitors. YkuE was purified from overproducing cells of the native host B. subtilis as described under “Experimental Procedures.” SDS-PAGE analysis of the YkuE-StrepII protein (∼35 kDa) was performed with a freshly purified sample obtained directly after elution from the Strep-Tactin column (lane 1), or with samples obtained after 20 h of incubation at 4 °C in the presence of either 1 mm N-ethylmaleimide (lane 2), 1 mm phenylmethylsulfonyl fluoride (lane 3), 10 mm EDTA (lane 4), or 1 mm EDTA (lane 5). Degradation bands observed upon protein incubation in buffered solution were identified by mass spectrometry to be part of the original full-length protein.

FIGURE 7.

Enzyme kinetics and observed catalytic efficiencies of YkuE under different pH conditions. A, freshly purified YkuE was used under restored conditions of Mn(II) binding for kinetic analysis. The monophosphoester compound p-nitrophenyl phosphate was used as a substrate, and the assays were conducted under varying pH conditions (between pH 6.5 and 9.5) at 25 °C. The formation of p-nitrophenol was monitored spectrophotometrically, and the obtained data were corrected for spontaneous background hydrolysis examined in nonenzymatic control reactions. Final data were plotted and analyzed by Michaelis-Menten-type nonlinear regression. The kinetic parameters for all pH-dependent reaction series are given in supplemental Table 3. B, obtained catalytic efficiencies were plotted versus their cognate pH to determine the optimum catalytic activity and the pK_a of the water nucleophile for YkuE-dependent hydrolysis. Because of the strongly pH-dependent k_cat values, the catalytic efficiencies vary over 1 order of magnitude within the tested pH range and show an optimum peak at around pH 9.0. The pK_a of the hydrated metal center is ∼7.80, revealing the strong activation potential of the binuclear catalytic site of YkuE.

FIGURE 8.

YkuE active site model. The proposed binuclear metal reaction center of YkuE is shown, and amino acid residues are indicated that are predicted to act as ligand donors at the metal cofactor site. Predictions were made upon comparison with various metallophosphoesterase consensus sequences, and their metal-binding motifs were based on protein database information (UniProt, NCBI). Putative metal selectivities at the distinct binding sites are further suggested according to the experimentally found dominant metal species associated with natively purified YkuE and based on known metal-ligand complex stabilities for varied N/O donor sites. The core processes of the proposed catalytic mechanism of YkuE are indicated by interaction of a substrate phosphoryl moiety with the bi-metal cofactor site, which also activates water for nucleophilic attack and subsequent hydrolysis of the currently not further specified phosphoester substrate compound(s).