Abstract
The rice pathogen Burkholderia glumae uses amino acids as a principal carbon source and thus produces ammonia in amino acid-rich culture medium such as Luria–Bertani (LB) broth. To counteract ammonia-mediated environmental alkaline toxicity, the bacterium produces a public good, oxalate, in a quorum sensing (QS)-dependent manner. QS mutants of B. glumae experience alkaline toxicity and may undergo cell death at the stationary phase when grown in LB medium. Here, we show that the cell-death processes of QS mutants due to alkaline environmental conditions are similar to the apoptosis-like cell death reported in other bacteria. Staining QS mutants with bis-(1,3-dibutylbarbituric acid)-trimethine oxonol revealed membrane depolarization. CellROX™ staining showed excessive generation of reactive oxygen species (ROS) in QS mutants. The expression of genes encoding HNH endonuclease (BGLU_1G15690), oligoribonuclease (BGLU_1G09120), ribonuclease E (BGLU_1G09400), and Hu-beta (BGLU_1G13530) was significantly elevated in QS mutants compared to that in wild-type BGR1, consistent with the degradation of cellular materials as observed under transmission electron microscopy (TEM). A homeostatic neutral pH was not attainable by QS mutants grown in LB broth or by wild-type BGR1 grown in an artificially amended alkaline environment. At an artificially adjusted alkaline pH, wild-type BGR1 underwent apoptosis-like cell death similar to that observed in QS mutants. These results show that environmental alkaline stress interferes with homeostatic neutral cellular pH, induces membrane depolarization, and causes apoptosis-like cell death in B. glumae.
Highlights
Bacteria have survival mechanisms to adapt to stressful conditions deviating from their niche conditions
Our findings revealed that B. glumae is inherently sensitive to alkaline environmental pH, and the mechanism underlying alkaline stress-induced cell death is similar to that by which apoptosis-like cell death occurs in other bacteria
To determine whether cell death in quorum sensing (QS) mutants at alkaline environmental pH is similar to the known apoptosis-like cell death in other bacteria, we first observed the cellular contents of two QS mutants, BGS2 (BGR1 tofI::Ω) and BGS9 (BGR1 qsmR::Ω), using transmission electron microscopy (TEM) during the stationary phase
Summary
Bacteria have survival mechanisms to adapt to stressful conditions deviating from their niche conditions. To cope with a stressful environment, bacteria use specific molecular and metabolic mechanisms. Bacterial cells reduce their size and increase the expression of specific genes to maintain continuous growth and survival (Ishihama, 1997; Nyström, 2004). In a highly osmotic external environment, bacteria possess several strategies for adjusting their cellular osmolarity through the import of ions and the synthesis of compatible solutes (Imhoff, 1986; Wood, 2006). Because pH is a powerful stress factor that can affect a variety of biological molecules, bacteria must maintain a cellular pH in the neutral range. The survival mechanisms of bacteria in stressful environments are well understood to date (Aertsen and Michiels, 2004; Justice et al, 2008). The molecular and physiological details of bacterial cell death under non-permissible conditions are less characterized
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