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Unbalanced membrane phospholipid compositions affect transcriptional expression of certain regulatory genes in
K Inoue1, H Matsuzaki, K Matsumoto
1Department of Biochemistry and Molecular Biology, Saitama University, Urawa, Japan.
Abstract:
The amount of porin protein OmpF in the outer membrane of Escherichia coli was reduced to one-third by the pgsA3 mutation that diminishes the amount of phosphatidylglycerol and cardiolipin in the membrane, whereas a cls (cardiolipin synthase) null mutation had no effect. Osmoregulation of OmpF was functional in the pgsA3 mutant. As assessed by the beta-galactosidase activities of lacZ fusions, the ompF expression was not reduced at the transcriptional level but was reduced about threefold at the posttranscriptional level by pgsA3. This reduction was mostly restored by a micF null mutation, and the micF RNA that inhibits the ompF mRNA translation was present 1.3 to 1.4 times more in the pgsA3 mutant, as assayed by RNase protection and Northern blot analyses. Elevation of the level of micF RNA was not restricted to acidic-phospholipid deficiency: OmpF was hardly detected and micF RNA was present 2.7 to 2.8 times more in a pssA null mutant that lacked phosphatidylethanolamine. Other common phenotypes of pgsA3 and pssA null mutants, reduced rates of cell growth and phospholipid synthesis, were not the cause of micF activation. Salicylate, which activates micF expression and inhibits cell motility, did not repress the flagellar master operon. These results imply that an unbalanced phospholipid composition, rather than a decrease or increase in the amount of specific phospholipid species, induces a phospholipid-specific stress signal to which certain regulatory genes respond positively or negatively according to their intrinsic mechanisms.
Insights
Escherichia coli phospholipid mutants show altered OmpF porin levels due to increased micF RNA. This suggests a phospholipid-specific stress response impacts gene regulation.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The outer membrane protein OmpF in Escherichia coli is crucial for nutrient uptake and is subject to complex regulation.
- Phospholipids are essential components of bacterial membranes, influencing protein function and cellular processes.
- Previous studies indicated a link between membrane lipid composition and porin expression, but the underlying mechanisms were unclear.
Purpose of the Study:
- To investigate the impact of altered phospholipid synthesis on OmpF porin levels in Escherichia coli.
- To elucidate the regulatory mechanisms, particularly the role of micF RNA, involved in phospholipid-mediated OmpF regulation.
- To determine if phospholipid imbalance triggers a specific stress response affecting gene expression.
Main Methods:
- Utilized pgsA3 and cls null mutations to alter membrane phospholipid content (phosphatidylglycerol, cardiolipin).
- Assessed OmpF protein levels and ompF gene expression using beta-galactosidase assays and Western blotting.
- Quantified micF RNA levels via RNase protection assays and Northern blot analyses.
- Investigated the role of micF in OmpF regulation through null mutations.
Main Results:
- The pgsA3 mutation, reducing phosphatidylglycerol and cardiolipin, decreased OmpF protein levels threefold at the posttranscriptional level, without affecting transcription.
- A micF null mutation largely restored OmpF levels in the pgsA3 mutant, accompanied by a 1.3-1.4 fold increase in micF RNA.
- A pssA null mutant lacking phosphatidylethanolamine also showed reduced OmpF and elevated micF RNA, indicating the effect is not limited to acidic phospholipids.
- Cell growth rates and phospholipid synthesis rates were not the direct cause of micF activation.
Conclusions:
- Altered phospholipid composition, rather than specific lipid levels, triggers a phospholipid-specific stress signal in Escherichia coli.
- This stress signal activates regulatory genes like micF, which in turn posttranscriptionally represses ompF mRNA translation.
- The findings reveal a novel regulatory pathway linking membrane lipid homeostasis to porin expression and cellular adaptation.