Lon Protease Removes Excess Signal Recognition Particle Protein in Escherichia coli.

Correct targeting of membrane proteins is essential for membrane integrity, cell physiology, and viability. Cotranslational targeting depends on the universally conserved signal recognition particle (SRP), which is a ribonucleoprotein complex comprised of the protein component Ffh and the 4.5S RNA in Escherichia coli About 25 years ago it was reported that Ffh is an unstable protein, but the underlying mechanism has never been explored. Here, we show that Lon is the primary protease responsible for adjusting the cellular Ffh level. When overproduced, Ffh is particularly prone to degradation during transition from exponential to stationary growth and the cellular Ffh amount is lowest in stationary phase. The Ffh protein consists of two domains, the NG domain, responsible for GTP hydrolysis and docking to the membrane receptor FtsY, and the RNA-binding M domain. We find that the NG domain alone is stable, whereas the isolated M domain is degraded. Consistent with the importance of Lon in this process, the M domain confers synthetic lethality to the lon mutant. The Ffh homolog from the model plant Arabidopsis thaliana, which forms a protein-protein complex rather than a protein-RNA complex, is stable, suggesting that the RNA-binding ability residing in the M domain of E. coli Ffh is important for proteolysis. Our results support a model in which excess Ffh not bound to 4.5S RNA is subjected to proteolysis until an appropriate Ffh concentration is reached. The differential proteolysis adjusts Ffh levels to the cellular demand and maintains cotranslational protein transport and membrane integrity.IMPORTANCE Since one-third of all bacterial proteins reside outside the cytoplasm, protein targeting to the appropriate address is an essential process. Cotranslational targeting to the membrane relies on the signal recognition particle (SRP), which is a protein-RNA complex in bacteria. We report that the protein component Ffh is a substrate of the Lon protease. Regulated proteolysis of Ffh provides a simple mechanism to adjust the concentration of the essential protein to the cellular demand. This is important because elevated or depleted SRP levels negatively impact protein targeting and bacterial fitness.

The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens.

Riboregulation involving regulatory RNAs, RNA chaperones, and ribonucleases is fundamental for the rapid adaptation of gene expression to changing environmental conditions. The gene coding for the RNase YbeY belongs to the minimal prokaryotic genome set and has a profound impact on physiology in a wide range of bacteria. Here, we show that the Agrobacterium tumefaciensybeY gene is not essential. Deletion of the gene in the plant pathogen reduced growth, motility, and stress tolerance. Most interestingly, YbeY is crucial for A. tumefaciens-mediated T-DNA transfer and tumor formation. Comparative proteomics by using isobaric tags for relative and absolute quantitation (iTRAQ) revealed dysregulation of 59 proteins, many of which have previously been found to be dependent on the RNA chaperone Hfq. YbeY and Hfq have opposing effects on production of these proteins. Accumulation of a 16S rRNA precursor in the ybeY mutant suggests that A. tumefaciens YbeY is involved in rRNA processing. RNA coimmunoprecipitation-sequencing (RIP-Seq) showed binding of YbeY to the region immediately upstream of the 16S rRNA. Purified YbeY is an oligomer with RNase activity. It does not physically interact with Hfq and thus plays a partially overlapping but distinct role in the riboregulatory network of the plant pathogen.IMPORTANCE Although ybeY gene belongs to the universal bacterial core genome, its biological function is incompletely understood. Here, we show that YbeY is critical for fitness and host-microbe interaction in the plant pathogen Agrobacterium tumefaciens Consistent with the reported endoribonuclease activity of YbeY, A. tumefaciens YbeY acts as a RNase involved in maturation of 16S rRNA. This report adds a worldwide plant pathogen and natural genetic engineer of plants to the growing list of bacteria that require the conserved YbeY protein for host-microbe interaction.

Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) including Variant Analysis by Mass Spectrometry in Placental Tissue.

Among neonates, tested positive for SARS-CoV-2, the majority of infections occur through postpartum transmission. Only few reports describe intrauterine or intrapartum SARS-CoV-2 infections in newborns. To understand the route of transmission, detection of the virus or virus nucleic acid in the placenta and amniotic tissue are of special interest. Current methods to detect SARS-CoV-2 in placental tissue are immunohistochemistry, electron microscopy, in-situ hybridization, polymerase chain reaction (PCR) and next-generation sequencing. Recently, we described an alternative method for the detection of viral ribonucleic acid (RNA), by combination of reverse transcriptase-PCR and mass spectrometry (MS) in oropharyngeal and oral swabs. In this report, we could detect SARS-CoV-2 in formal-fixed and paraffin-embedded (FFPE) placental and amniotic tissue by multiplex RT-PCR MS. Additionally, we could identify the British variant (B.1.1.7) of the virus in this tissue by the same methodology. Combination of RT-PCR with MS is a fast and easy method to detect SARS-CoV-2 viral RNA, including specific variants in FFPE tissue.