| Research Abstract |
A large number of Escherichia coli cells become viable but non-culturable (VBNC) at early stationary phase, most of which are directed to lyses in cells with an enhanced active sigma E level, eliminating VBNC cells. The lysis process, however, has not been elucidated except for the evidence of reduction of outer membrane proteins, OmpA, OmpC and OmpW. Recently, it was discovered that micA and rybB encoding sRNA, which are involved in the negative regulation of OmpA, OmpC, and OmpW, are regulated by sigma E. The aim of this study is to decipher the cascade of the sigma E-dependent programmed cell death on the assumption that micA and rybB as a small RNA are involved in the cascade. Especially, we focused on the trigger as a signal to induce the lysis process and the cascade from repression of omp expression to lysis.
The defective strain of katE, encoding catalase, exhibited a s sigma E-dependent cell lysis phenotype at early stationary phase. The level of intracellular reactive oxygen s
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pecies (ROS) became maximal at the transition period from exponential to stationary phases, and VBNC cells gradually increased after that period. In the wild-type strain, both sodA, encoding superoxide dismutase, and katE plasmid clones almost completely suppressed cell lysis, largely reduced ROS, and substantially increased culturable cells. These results suggest that oxidative stress is a major factor responsible for VBNC cell formation and cell lysis at the early stationary phase. Therefore, these results suggest that oxidative stress (intracellular ROS) is a signal to give rise to VBNC cells, which are in turn lysed by a sigma E-dependent process. Experiments with mutants or plasmid clones of micA, rybB, ompA, ompC and ompW and effect of Mg^<2+> suggest that the cell lysis proceeds in the cascade of sigma E→expression of micA and rybB→reduction of Omp proteins→disintegration of the outer membrane. These results with others presented reveal a novel function of sRNA to control the cell lysis. Significant characteristics of cell lysis, accompanied by a severe reduction in the levels of periplasmic OMP-folding factor (PpiD), were observed in a mutant of rseA encoding anti-sigma E. The cell-lysis phenotype of the mutant was suppressed by either rseA or ppiD plasmids. Thus, increase in the ratio of free of in rseA mutants with a concomitant reduction in PpiD levels can account for sigma E-dependent lysis in concert with a potential role of small RNAs on the lysis process.
Altogether, intracellular ROS is accumulated at the beginning of stationary phase and VBNC cells increase to cause cell lysis, sigma E-dependent PCD. In the cascade of the sigma E-dependent PCD, the accumulation of active sigma E molecules up-regulates sRNA of micA and rybB to repress the expression of Omp genes (ompA, ompC and ompW) and somehow decreases PpiD. Their both negative effects decrease OmpA, OmpC, OmpW, which weaken outer membrane structure to cause cell lysis. Less
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