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 factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative factors are the extracytoplasmic function (ECF) factors. In this paper, we demonstrate that the expression of the ECF factor 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 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 V . Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for Vdependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF 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 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.The majority of genes in actively growing bacteria are transcribed by RNA polymerase using the general "housekeeping" factor 70 . Bacteria often utilize alternative factors to regulate subsets of genes required for specific environmental conditions (18). The largest group of these alternative factors are the extracytoplasmic function (ECF) factors (18, 39). ECF factors represent the "third pillar" of bacterial signal transduction and are often involved in response to extracytoplasmic stress (18,39). ECF factors are members of the 70 family of factors and are characterized by the presence of only two regions of 70 , regions 2 and 4.2 (18). Bacillus subtilis encodes seven known ECF factors (18). ECF factors are often required for their own transcription; thus, the expression of an ECF factor promoter is often indicative of activity of the ECF factor (18, 39). The signals which induce the activity of several ECF factors are known. For instance, the expression of sigW is induced by antimicrobial peptides and pH change (6, 9, 14, 32, 45), while sigM expression is induced by inhibitors of cell wall biosynthesis, heat shock, paraquat, and ethanol stress (12,40). Like sigM, sigX is induced by in...

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

“…lmo2473 encodes an uncharacterized protein that has been hypothesized to function in the synthesis of peptidoglycan precursors (36), and lmo2768 encodes an uncharacterized membrane protein with an ABC transporter domain. lmo0540 (here referred to as pbpX) is the homolog of pbpX in B. subtilis, where it contributes to lysozyme resistance (37). pbpX encodes a ␤-lactamase domain; however, it has been reported that in Mycobacterium smegmatis, PbpX does not contribute to ␤-lactam antibiotic resistance.…”

“…subtilis also contains pbpX, oatA, and the genes for multiple peptidoglycan deacetylases (13,37,61), yet B. subtilis is not a pathogen and is significantly more sensitive to lysozyme than L. monocytogenes. The MIC of lysozyme is 6 g/ml for WT B. subtilis (13) and 2,000 g/ml for L. monocytogenes (data not shown).…”

Effect of CMC and MCC as Sole Carbon Sources on Cellulase Activity and eglS Gene Expression in Three Bacillus subtilis Strains Isolated from Corn Stover

“…The oligonucleotides used for eglS expression (Fwd-GTTCACACGGATTGCAATGG, rev-TACATCGCTGCACGGAAAAC) were designed with Primer3 plus (Untergasser et al 2012) using the eglS gene from B. subtilis 168 (Barbe et al 2009). The rpoB gene was selected as a reference gene for normalization (Ho et al 2011). In order to evaluate the reproducibility of the reaction, a calibration curve was prepared using 1000, 100, 10, 1, and 0.1 ng of cDNA from a control sample and 200 nmol/L of each oligonucleotide from the reference gene according to Nolan et al (2006).…”

Activation without Proteolysis of Anti-<i>σ</i> Factor RsiV of the Extracytoplasmic Function <i>σ</i> Factor <i>σ</i><sup>V</sup> in a Glucolipid-Deficient Mutant of <i>Bacillus subtilis</i>

“…The genes for the σ factors and the anti-σ factors form operons whose transcription is directed by the cognate σ factors; the activities of the promoters for ECF σ factor genes are thus indicative of the activities of the respective ECF σ factors [1] [2] [3]. Among these ECF σ factors, σ V primarily responds to lysozyme [4]. It is activated by stepwise proteolytic destruction of the anti-σ factor RsiV via regulated intramembrane proteolysis (RIP) [5].…”