Kangseok Lee

RNase G complementation of rne null mutation identifies functional interrelationships with RNase E in Escherichia coli

The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity. Whereas the absence of Rne normally is lethal, we show here that E. coli bacteria that lack the rne gene can be made viable by overexpression of Rng. Rng‐complemented cells accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase P (i.e. M1 RNA), indicating that normal processing of these Rne‐cleaved RNAs was not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from precursors by RNase G cleavage in vitro. Using DNA microarrays containing 4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose steady‐state level was affected by Rne, Rng or the N‐terminal catalytic domain of RNase E. Most transcript species affected by RNase E deficiency were also elevated in an rne deletion mutant complemented by Rng. However, approximately 100 mRNAs that accumulated in Rne‐deficient cells were decreased by rng‐complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energy‐generating pathways or in the synthesis or degradation of macromolecules.

RraA. a protein inhibitor of RNase E activity that globally modulates RNA abundance in E. coli

Ribonuclease E (RNase E) has a key role in mRNA degradation and the processing of catalytic and structural RNAs in E. coli. We report the discovery of an evolutionarily conserved 17.4 kDa protein, here named RraA (regulator of ribonuclease activity A) that binds to RNase E and inhibits RNase E endonucleolytic cleavages without altering cleavage site specificity or interacting detectably with substrate RNAs. Overexpression of RraA circumvents the effects of an autoregulatory mechanism that normally maintains the RNase E cellular level within a narrow range, resulting in the genome-wide accumulation of RNase E-targeted transcripts. While not required for RraA action, the C-terminal RNase E region that serves as a scaffold for formation of a multiprotein degradosome complex modulates the inhibition of RNase E catalytic activity by RraA. Our results reveal a possible mechanism for the dynamic regulation of RNA decay and processing by inhibitory RNase binding proteins.

Differential immunostimulatory effects of Gram-positive bacteria due to their lipoteichoic acids.

Lipoteichoic acid (LTA) is a major immunostimulating component in the cell wall of Gram-positive bacteria as lipopolysaccharide of Gram-negative bacteria. However, LTA is expressed on not only pathogenic but also nonpathogenic Gram-positive bacteria. In order to examine whether the immunostimulating potentials of Gram-positive bacteria are correlated with their LTAs, we prepared highly pure LTAs from Staphylococcus aureus (pathogenic), Bacillus subtilis (non-pathogenic), or Lactobacillus plantarum (beneficial). When a murine macrophage cell-line, RAW 264.7, was stimulated with heat-killed bacteria, both S. aureus and B. subtilis induced nitric oxide (NO) production in a dose-dependent manner while L. plantarum showed a minimal induction. Interestingly, purified LTAs from S. aureus and B. subtilis, but not from L. plantarum, were able to induce the production of NO. The differential inflammatory potentials of LTAs coincided with their abilities to activate Toll-like receptor 2 (TLR2), which is known to recognize Gram-positive bacteria and LTA, and transcription factors NF-kappaB and AP-1. Similar results were obtained with the expression of cytokines related to inflammation by RAW 264.7 and human peripheral blood mononuclear cells as well. The ability of LTA to induce TNF-alpha and NO production was abolished when the LTAs were treated with 0.2 N NaOH. Collectively, we suggest that the immunostimulating potentials of Gram-positive bacteria differ due to their LTAs with differential potencies in the stimulation of TLR2.

A Streptomyces coelicolor functional orthologue of Escherichia coli RNase E shows shuffling of catalytic and PNPase‐binding domains

Previous work has detected an RNase E‐like endoribonucleolytic activity in cell extracts obtained from Streptomyces. Here, we identify a Streptomyces coelicolor gene, rns, encoding a 140 kDa protein (RNase ES) that shows endoribonucleolytic cleavage specificity characteristic of RNase E, confers viability on and allows propagation of Escherichia coli cells lacking RNase E and accomplishes RNase E‐like regulation of plasmid copy number in E. coli. However, notwithstanding its complementation of rne‐deleted E. coli, RNase ES did not accurately process 9S rRNA from E. coli. Additionally, whereas RNase E is normally required for E. coli survival, rns is not an essential gene in S. coelicolor. Deletion analysis mapped the   catalytic   domain   of   RNase   ES   near   its   centre and showed that regions located near the RNase ES termini interact with an S. coelicolor homologue of polynucleotide phosphorylase (PNPase) – a major component of E. coli RNase E‐based degradosomes. The interacting arginine‐ and proline‐rich segments resemble the C‐terminally located degradosome scaffold region of E. coli RNase E. Our results indicate that RNase ES is a structurally shuffled RNase E homologue showing evolutionary conservation of functional RNase E‐like enzymatic activity, and suggest the existence of degradosome‐like complexes in Gram‐positive bacteria.

Differential modulation of E. coli mRNA abundance by inhibitory proteins that alter the composition of the degradosome

In Escherichia coli the initial step in the processing or decay of many messenger and structural RNAs is mediated by the endonuclease RNase E, which forms the core of a large RNA‐catalysis machine termed the degradosome. Previous experiments have identified a protein that globally modulates RNA abundance by binding to RNase E and regulating its endonucleolytic activity. Here we report the discovery of RraB, which interacts with a different site on RNase E and interferes with cleavage of a different set of transcripts. We show that expression of RraA or RraB in vivo is accompanied by dramatic, distinct, and inhibitor‐specific changes in degradosome composition – and that these are in turn associated with alterations in RNA decay and global transcript abundance profiles that are dissimilar to the profile observed during simple RNase E deficiency. Our results reveal the existence of endonuclease binding proteins that modulate the remodelling of degradosome composition in bacteria and argue that such degradosome remodelling is a mechanism for the differential regulation of RNA cleavages in E. coli.

Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.

FOXL2 Interacts with Steroidogenic Factor-1 (SF-1) and Represses SF-1-Induced CYP17 Transcription in Granulosa Cells

Mutations in FOXL2 are responsible for blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) type I, in which affected women exhibit premature ovarian failure. FOXL2-null mice showed defects in granulosa cell development during folliculogenesis. We screened a rat ovarian yeast two-hybrid cDNA library to identify FOXL2-interacting proteins and found steroidogenic factor-1 (SF-1). Here, we show that human FOXL2 and SF-1 proteins interact in human granulosa cells and that FOXL2 negatively regulates the transcriptional activation of a steroidogenic enzyme, CYP17, by SF-1. Furthermore, FOXL2 mutants found in blepharophimosis-ptosis-epicanthus inversus syndrome type I patients lost the ability to repress CYP17 induction mediated by SF-1. Chromatin immunoprecipitation and EMSA results further revealed that FOXL2 inhibited the binding of SF-1 to the CYP17 promoter, whereas the FOXL2 mutants failed to block this interaction. Therefore, this study identifies a novel regulatory role for FOXL2 on a key steroidogenic enzyme and provides a possible mechanism by which mutations in FOXL2 disrupt normal ovarian follicle development.

Funnel-like Hexameric Assembly of the Periplasmic Adapter Protein in the Tripartite Multidrug Efflux Pump in Gram-negative Bacteria

Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. The Escherichia coli AcrAB-TolC pump is the principal multidrug exporter that confers intrinsic drug tolerance to the bacteria. The inner membrane transporter AcrB requires the outer membrane factor TolC and the periplasmic adapter protein AcrA. However, it remains ambiguous how the three proteins are assembled. In this study, a hexameric model of the adapter protein was generated based on the propensity for trimerization of a dimeric unit, and this model was further validated by presenting its channel-forming property that determines the substrate specificity. Genetic, in vitro complementation, and electron microscopic studies provided evidence for the binding of the hexameric adapter protein to the outer membrane factor in an intermeshing cogwheel manner. Structural analyses suggested that the adapter covers the periplasmic region of the inner membrane transporter. Taken together, we propose an adapter bridging model for the assembly of the tripartite pump, where the adapter protein provides a bridging channel and induces the channel opening of the outer membrane factor in the intermeshing tip-to-tip manner.

Lipoteichoic Acid Partially Contributes to the Inflammatory Responses to Enterococcus faecalis

Enterococcus faecalis, a pathogenic gram-positive bacterium, is closely related to refractory apical periodontitis. Because lipoteichoic acid (LTA) is considered a major virulence factor of gram-positive bacteria, in the present study, highly pure LTA from E. faecalis was prepared, and its ability to stimulate murine macrophages was investigated in comparison with those of the killed whole cells. Upon exposure to E. faecalis LTA, RAW 264.7 (a murine macrophage cell line) produced a significantly (p < 0.05) high level of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in a concentration-dependent manner. It is to note that the LTA was able to stimulate Toll-like receptor 2 (TLR2) but not TLR4. Concomitantly, LTA enhanced the DNA-binding activity of a transcription factor, nuclear factor-kappa B (NF-kappaB), which plays an important role in the transcriptional activation of genes encoding inflammatory mediators. In contrast, heat-killed E. faecalis stimulated both TLR2 and TLR4, whereas the killed E. faecalis whole cells induced significant (p < 0.05) levels of TNF-alpha and NO in RAW 264.7 cells as their LTA did. These results suggest that LTA partially contributes to E. faecalis-induced inflammatory responses.

Escherichia coli ribonuclease III activity is downregulated by osmotic stress: consequences for the degradation of bdm mRNA in biofilm formation

During the course of experiments aimed at identifying genes with ribonuclease III (RNase III)‐dependent expression in Escherichia coli, we found that steady state levels of bdm mRNA were dependent on cellular concentrations of RNase III. The half‐lives of adventitiously overexpressed bdm mRNA and the activities of a transcriptional bdm‘–’cat fusion were observed to be dependent on cellular concentrations of RNase III, indicating the existence of cis‐acting elements in bdm mRNA responsive to RNase III. In vitro and in vivo cleavage analyses of bdm mRNA identified two RNase III cleavage motifs, one in the 5′‐untranslated region and the other in the coding region of bdm mRNA, and indicated that RNase III cleavages in the coding region constitute a rate‐determining step for bdm mRNA degradation. We also discovered that downregulation of the ribonucleolytic activity of RNase III is required for the sustained elevation of RcsB‐induced bdm mRNA levels during osmotic stress and that cells overexpressing bdm form biofilms more efficiently. These findings indicate that the Rcs signalling system has an additional regulatory pathway that functions to modulate bdm expression and consequently, adapt E. coli cells to osmotic stress.