The Bacillus subtilis extracytoplasmic function σ factor σ(V) is induced by lysozyme and provides resistance to lysozyme

Abstract

Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative σ factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative σ factors are the extracytoplasmic function (ECF) σ factors. In this paper, we demonstrate that the expression of the ECF σ factor σ(V) in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that σ(V) is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon σ(V). Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for σ(V)-dependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF σ factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF σ factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF σ factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme.

Fig. 1.

Expression of sigV is specifically induced by lysozyme stress. The strains contained P_sigV-lacZ and were either wild type (CDE1546) or sigV mutant (CDE1547). Cells were grown to an OD₆₀₀ of 2, placed in LB top agar containing X-Gal, spread on solid LB medium plus X-Gal, and allowed to solidify. Filter disks containing phosphomycin (Pho; 10 μl of 5-mg/ml concentration), cycloserine (Cyc; 10 μl of a 5-mg/ml concentration), tunicamycin (Tun; 10 μl of a 100-μg/ml concentration), nisin (Nis; 10 μl of a 50-mg/ml concentration), ramoplanin (Ram; 10 μl of a 1-mg/ml concentration), vancomycin (Van; 10 μl of a 1-mg/ml concentration), cefoxitin (Cef; 10 μl of a 100-μg/ml concentration), ampicillin (Amp; 10 μl of a 1-mg/ml concentration), bacitracin (Bac; 10 μl of a 1-mg/ml concentration), hen egg white lysozyme (Lys; 10 μl of a 20-mg/ml concentration), polymyxin B (Poly; 10 μl of a 100-mg/ml concentration), EDTA (10 μl of a 0.5 M concentration), deoxycholate (Deo; 10 μl of a 10% concentration), or proteinase K (Pro K; 10 μl of a 1-mg/ml concentration) were then placed on the top agar and incubated for 16 h at 37°C.

Fig. 2.

sigV is the only lysozyme-induced σ factor. The strains used contained an ECF σ factor promoter-lacZ fusion integrated at amyE and were otherwise wild type. Shown are results obtained with sigV (amyE:: P_sigV-lacZ CDE702), sigM (amyE::P_sigV-lacZ CDE680), sigX (amyE:: P_sigX-lacZ CDE705), sigV (amyE::P_sigV-lacZ CDE1547), sigY (amyE::P_sigY-lacZ CDE708), sigZ (amyE::P_sigZ-lacZ CDE711), ylaC (amyE::P_sigV-lacZ CDE714), and sigW (amyE::P_sigW-lacZ CDE717) mutant cells. Cells were grown to an OD₆₀₀ of 1.0 and spotted onto plates containing either LB or LB plus 2.5 μg/ml hen egg white lysozyme. The plates were incubated at 37°C for 6 h. Cells were recovered from the plates and resuspended in Z buffer, and β-galactosidase activities were determined as described in Materials and Methods. The results shown are averages of three experiments, and the error bars denote standard deviations. The data plotted are fold induction by lysozyme (lysozyme β-galactosidase activity/no-lysozyme β-galactosidase activity).

Fig. 3.

Roles of σ^V and oatA in resistance to lysozyme. The MIC of lysozyme was determined for wild-type (WT; strain PY79) and sigV (CDE1712; ΔsigV), oatA (CDE1581; ΔoatA::kan), and sigV-oatA (CDE1571; ΔsigV-rsiV-oatA::kan) mutant strains without and with an IPTG-inducible copy of oatA⁺ (amyE::P_hs-oatA⁺), as follows: wild-type oatA⁺ (CDE1713; amyE::P_hs-oatA⁺) sigV oatA⁺ (CDE1713; amyE::P_hs-oatA⁺ ΔsigV), oatA-oatA⁺ (CDE1581; amyE::P_hs-oatA⁺ ΔoatA::kan), and sigV-oatA oatA⁺ (CDE1571; amyE::P_hs-oatA⁺ ΔsigV-rsiV-oatA::kan). The MIC is defined as no growth (OD₆₀₀ less than 0.05) after 16 h at 37°C in a 96-well plate. The MIC experiments were performed in technical triplicates and repeated on different days. The data shown are from one representative experiment. The MIC varied slightly between days or experiments; however, the fold changes always remained the same.

Fig. 4.

oatA is part of the sigV operon and is lysozyme inducible. (A) sigV is in a four-gene operon. The top portion shows the putative sigV operon structure. Capital letters represent PCR primer pairs (A, CDEP273-CDEP274; B, CDEP1015-CDEP978; C, CDEP1011-CDEP1012; D, CDEP1013-CDEP1014) in which the mRNA from the same samples had (+) or had not (−) been treated with reverse transcriptase (RT) as described in Materials and Methods. The DNA was then separated on a 1.5% agarose gel. (B) Expression of oatA is induced by lysozyme stress. The oligonucleotides used in the qRT-PCR for rsiV (CDEP1107 and CDEP1108) and oatA (CDEP1021 and CDEP1022) are listed in Table S1 in the supplemental material. RNA levels were corrected to the standard rpoB (CDEP1017 and CDEP1018). Shown are the mean ± standard deviation of the relative change in RNA levels 15 min following exposure to a subinhibitory concentration of lysozyme (1.25 μg/ml hen egg white lysozyme). The qRT experiments were performed in technical triplicates and repeated on different days. The data shown are from one representative experiment. wt, wild type.

Fig. 5.

The combination of ECF σ factors mutations increases lysozyme sensitivity. The MIC of lysozyme was determined for the wild type (WT; strain PY79); for sigV (CDE1712; ΔsigV), sigW (CDE431; sigW::kan), sigX (CDE1718; sigX::spc), sigM (CDE1717; sigM::kan), sigY (CDE1719; sigY::kan), sigZ (CDE1720; sigZ::kan), and ylaC (CDE1721; ylaC::kan) single ECF σ factor mutant strains; for sigV sigW (CDE1723; ΔsigV sigW::kan), sigV sigX (CDE1727; ΔsigV sigX::spc), sigV sigM (CDE1724; ΔsigV sigM::kan), sigV sigY (CDE1722; ΔsigV sigY::kan), sigV sigZ (CDE1725; ΔsigV sigZ::kan), sigV ylaC (CDE1726; ΔsigV ylaC::kan) and sigM sigX (CDE1779; sigM::kan sigX::spc) ECF σ factor double mutant strains; and for a sigM sigV sigX (CDE1778; ΔsigV sigM::kan sigX::spc) ECF σ factor triple mutant strain. The MIC was defined was no growth (OD₆₀₀ less than 0.05) after 16 h at 37°C in a 96-well plate. The MIC experiments were performed in technical triplicates and repeated on different days. The data shown are from one representative experiment. The MIC varied slightly between days or experiments; however, the fold changes always remained the same.

Fig. 6.

Two ECF σ factor-regulated genes, dltA and pbpX, are required for maximal lysozyme resistance. The MIC of lysozyme was determined for the wild type (WT; strain PY79) and sigV (CDE1712; ΔsigV), dltA (CDE1709; ΔdltA::kan), oatA (CDE1581; ΔoatA::kan), and pbpX (CDE1709; ΔpbpX::kan) mutant strains. The MIC was defined as no growth (OD₆₀₀ less than 0.05) after 16 h at 37°C in a 96-well plate. The MIC experiments were performed in technical triplicates and repeated on different days. The data shown are from one representative experiment. The MIC varied slightly between days or experiments; however, the fold changes always remained the same.