Membrane targeting of ribosomes and their release require distinct and separable functions of FtsY

The mechanism underlying the interaction of the Escherichia coli signal recognition particle (SRP) receptor FtsY with the cytoplasmic membrane is not fully understood. We investigated this issue by utilizing active (NG+1) and inactive (NG) mutants of FtsY. In solution, the mutants comparably bind and hydrolyze nucleotides and associate with SRP. In contrast, a major difference was observed in the cellular distribution of NG and NG+1. Unlike NG+1, which distributes almost as the wild-type receptor, the inactive NG mutant accumulates on the membrane, together with ribosomes and SRP. The results suggest that NG function is compromised only at a later stage of the targeting pathway and that despite their identical behavior in solution, the membrane-bound NG-SRP complex is less active than NG+1-SRP. This notion is strongly supported by the observation that lipids stimulate the GTPase activity of NG+1-SRP, whereas no stimulation is observed with NG-SRP. In conclusion, we propose that the SRP receptor has two distinct and separable roles in (i) mediating membrane targeting and docking of ribosomes and (ii) promoting their productive release from the docking site.     

Membrane Protein Biogenesis in Ffh- or FtsY-Depleted Escherichia coli

Background

The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically.

Methodology/Principal Findings

We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization.

Conclusions

Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane.     

Migratory birds as the vehicle of transmission of multi drug resistant extended spectrum β lactamase producing Escherichia fergusonii,an emerging zoonotic pathogen

The acquisition of multi-drug resistance (MDR) genes by pathogenic bacterial bugs and their dispersal to different food webs has become a silent pandemic. The multiplied use of different antibacterial therapeutics during COVID-19 pandemic has accelerated the process among emerging pathogens. Wild migratory birds play an important role in the spread of MDR pathogens and MDR gene flow due to the consumption of contaminated food and water. Escherichia fergusonii is an emerging pathogen of family Enterobacteriaceae and commonly causes disease in human and animals. The present study focused on the isolation of E. fergusonii from blood, saliva, and intestine of selected migratory birds of the Hazara Division. The sensitivity of isolated strains was assessed against ten different antibiotics. The isolation frequency of E. fergusonii was 69%. In blood samples, a high rate of resistance was observed against ceftriaxone (80%) followed by ampicillin (76%) whereas, in oral and intestinal samples, ceftriaxone resistant strains were 56% and 57% while ampicillin resistance was 49% and 52% respectively. The overall ceftriaxone and ampicillin-resistant cases in all three sample sources were 71% and 65% respectively. In comparison to oral and intestinal samples, high numbers of ceftriaxone-resistant strains were isolated from the blood of mallard while ampicillin-resistant strains were observed in blood samples of cattle egrets. 16S rRNA-based confirmed strains of E. fergusonii were processed for detection of CTX-M and TEM-1 gene through Polymerase chain reaction (PCR) after DNA extraction. Hundred percent ceftriaxone resistant isolates possessed CTX-M and all ampicillin-resistant strains harbored TEM-1 genes. Amplified products were sequenced by using the Sanger sequencing method and the resulted sequences were checked for similarity in the nucleotide Database through the BLAST program. TEM-1 gene showed 99% and the CTX-M gene showed 98% similar sequences in the Database. The 16S rRNA sequence and nucleotide sequences for TEM-1 and CTX-M genes were submitted to Gene Bank with accession numbers {"type":"entrez-nucleotide","attrs":{"text":"LC521304","term_id":"1806500528","term_text":"LC521304"}}LC521304, {"type":"entrez-nucleotide","attrs":{"text":"LC521306","term_id":"1806500530","term_text":"LC521306"}}LC521306, {"type":"entrez-nucleotide","attrs":{"text":"LC521307","term_id":"1806500532","term_text":"LC521307"}}LC521307 respectively. We posit to combat MDR gene flow among the bacterial pathogens across different geographical locations, regular surveillance of new zoonotic pathogens must be conducted.     

Withdrawn as duplicate: Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development

This article has been withdrawn due to a publisher error that caused the article to be duplicated. The definitive version of this article is published under DOI 10.1093/plcell/koab115.

This has been withdrawn as a duplicate     

The role of transmembrane domain III in the lactose permease of Escherichia coli.

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.     

Tumor necrosis factor alpha induces stronger cytotoxicity in ABCG2-overexpressing resistant breast cancer cells compared with their drug-sensitive parental line

Tumor necrosis factor alpha (TNF-α) has been reported to modulate the multidrug resistance (MDR) phenotype in vitro and in vivo. Multidrug-resistant cells overexpressing the ABCB1 transporter are more susceptible to inhibition of proliferation and induction of apoptosis by TNF-α than their drug-sensitive counterparts. This study was aimed to investigate TNF-α modulatory and antiproliferative effects on drug-resistant cells overexpressing ABCG2. The effects of TNF-α on viability and proliferation rate of MCF-7 breast cancer cells and their ABCG2-overexpressing sublines MCF-7/mitoxantrone (MX) cells were studied using dye exclusion assay, dimethylthiazolyl-2,5-diphenyl tetrazolium bromide technique, and flow cytometric analysis of cell cycle. TNF-α influence on MX accumulation was investigated by flow cytometry. ABCG2-overexpressing cells were more susceptible to antiproliferative and cytotoxic effects of TNF-α than their parental cells. TNF-α increased accumulation of MX in both parental and resistant cells. Higher sensitivity of MDR cells to TNF-α cytotoxicity would help in characterization of its complex modulatory effects on cancer cells and benefit us in designing new approaches to overcome MDR.     

Evidence for simultaneous binding of dissimilar substrates by the Escherichia coli multidrug transporter MdfA

The mechanism by which multidrug transporters interact with structurally unrelated substrates remains enigmatic. Based on transport competition experiments, photoaffinity labeling, and effects on enzymatic activities, it was proposed in the past that multidrug transporters can interact simultaneously with a number of dissimilar substrate molecules. To study this phenomenon, we applied a direct binding approach and transport assays using the Escherichia coli multidrug transporter MdfA, which exports both positively charged (e.g., tetraphenylphosphonium, TPP(+)), zwitterionic (e.g., ciprofloxacin), and neutral (e.g., chloramphenicol) drugs. The interaction of MdfA with various substrates was examined by direct binding assays with the purified transporter. The immobilized MdfA binds TPP(+) in a specific manner, and all the tested positively charged substrates inhibit TPP(+) binding. Surprisingly, although TPP(+) binding is not affected by zwitterionic substrates, the neutral substrate chloramphenicol stimulates TPP(+) binding by enhancing its affinity to MdfA. In contrast, transport competition assays show inhibition of TPP(+) transport by chloramphenicol. We suggest that MdfA binds TPP(+) and chloramphenicol simultaneously to distinct but interacting binding sites, and the interaction between these two substrates during transport is discussed.     

Survival of Staphylococcus aureus ST398 in the Human Nose after Artificial Inoculation

There is evidence that MRSA ST398 of animal origin is only capable of temporarily occupying the human nose, and it is therefore, often considered a poor human colonizer.We inoculated 16 healthy human volunteers with a mixture of the human MSSA strain 1036 (ST931, CC8) and the bovine MSSA strain 5062 (ST398, CC398), 7 weeks after a treatment with mupirocin and chlorhexidine-containing soap. Bacterial survival was studied by follow-up cultures over 21 days. The human strain 1036 was eliminated faster (median 14 days; range 2–21 days) than the bovine strain 5062 (median 21 days; range 7–21 days) but this difference was not significant (p = 0.065). The bacterial loads were significantly higher for the bovine strain on day 7 and day 21. 4/14 volunteers (28.6%) showed elimination of both strains within 21 days. Of the 10 remaining volunteers, 5 showed no differences in bacterial counts between both strains, and in the other 5 the ST398 strain far outnumbered the human S. aureus strain. Within the 21 days of follow-up, neither human strain 1036 nor bovine strain 5062 appeared to acquire or lose any mobile genetic elements. In conclusion, S. aureus ST398 strain 5062 is capable of adequately competing for a niche with a human strain and survives in the human nose for at least 21 days.     

Glucose-6-phosphate dehydrogenase deficiency in Tunisia: molecular data and phenotype-genotype association

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect. In this study, we aimed to perform a molecular investigation of G6PD deficiency in Tunisia and to associate clinical manifestations and the degree of deficiency with the genotype. A total of 161 Tunisian subjects of both sexes were screened by spectrophotometric assay for enzyme activity. Out of these, 54 unrelated subjects were selected for screening of the most frequent mutations in Tunisia by PCR/RFLP, followed by size-based separation of double-stranded fragments under non-denaturing conditions on a denaturing high performance liquid chromatography system. Of the 56 altered chromosomes examined, 75 % had the GdA^? mutation, 14.28 % showed the GdB^? mutation and no mutations were identified in 10.72 % of cases. Hemizygous males with GdA^? mutation were mostly of class III, while those with GdB^? mutation were mainly of class II. The principal clinical manifestation encountered was favism. Acute hemolytic crises induced by drugs or infections and neonatal jaundice were also noted. Less severe clinical features such as low back pain were present in heterozygous females and in one homozygous female. Asymptomatic individuals were in majority heterozygote females and strangely one hemizygous male. The spectrum of mutations seems to be homogeneous and similar to that of Mediterranean countries; nevertheless 10.72 % of cases remain with undetermined mutation thus suggesting a potential heterogeneity of the deficiency at the molecular level. On the other hand, we note a better association of the molecular defects with the severity of the deficiency than with clinical manifestations.     

Accumulation of endoplasmic membranes and novel membrane-bound ribosome-signal recognition particle receptor complexes in Escherichia coli

In Escherichia coli, ribosomes must interact with translocons on the membrane for the proper integration of newly synthesized membrane proteins, cotranslationally. Previous in vivo studies indicated that unlike the E. coli signal recognition particle (SRP), the SRP receptor FtsY is required for membrane targeting of ribosomes. Accordingly, a putative SRP-independent, FtsY-mediated ribosomal targeting pathway has been suggested (Herskovits, A.A., E.S. Bochkareva, and E. Bibi. 2000. Mol. Microbiol. 38:927-939). However, the nature of the early contact of ribosomes with the membrane, and the involvement of FtsY in this interaction are unknown. Here we show that in cells depleted of the SRP protein, Ffh or the translocon component SecE, the ribosomal targeting pathway is blocked downstream and unprecedented, membrane-bound FtsY-ribosomal complexes are captured. Concurrently, under these conditions, novel, ribosome-loaded intracellular membrane structures are formed. We propose that in the absence of a functional SRP or translocon, ribosomes remain jammed at their primary membrane docking site, whereas FtsY-dependent ribosomal targeting to the membrane continues. The accumulation of FtsY-ribosome complexes induces the formation of intracellular membranes needed for their quantitative accommodation. Our results with E. coli, in conjunction with recent observations made with the yeast Saccharomyces cerevisiae, raise the possibility that the SRP receptor-mediated formation of intracellular membrane networks is governed by evolutionarily conserved principles.