The <i>Escherichia coli</i> outer membrane β-barrel assembly machinery (Bam) crosstalks with the divisome

The BAM is a macromolecular machine responsible for the folding and the insertion of integral proteins into the outer membrane of diderm Gram-negative bacteria. In Escherichia coli, it consists of a transmembrane β-barrel subunit, BamA, and four outer membrane lipoproteins (BamB-E). Using BAM-specific antibodies, in E. coli cells, the complex is shown to localize in the lateral wall in foci. The machinery was shown to be enriched at midcell with specific cell cycle timing. The inhibition of septation by aztreonam did not alter the BAM midcell localization substantially. Furthermore, the absence of late cell division proteins at midcell did not impact BAM timing or localization. These results imply that the BAM enrichment at the site of constriction does not require an active cell division machinery. Expression of the Tre1 toxin, which impairs the FtsZ filamentation and therefore midcell localization, resulted in the complete loss of BAM midcell enrichment. A similar effect was observed for YidC, which is involved in the membrane insertion of cell division proteins in the inner membrane. The presence of the Z-ring is needed for preseptal peptidoglycan (PG) synthesis. As BAM was shown to be embedded in the PG layer, it is possible that BAM is inserted preferentially simultaneously with de novo PG synthesis to facilitate the insertion of OMPs in the newly synthesized outer membrane

Early encounters of a nascent membrane protein: specificity and timing of contacts inside and outside the ribosome

An unbiased photo–cross-linking approach was used to probe the “molecular path” of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome–nascent chain complex to the Sec–YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome

YidC and SecY mediate membrane insertion of a type I transmembrane domain

YidC has been identified recently as an evolutionary conserved factor that is involved in the integration of inner membrane proteins (IMPs) in Escherichia coli. The discovery of YidC has inspired the reevaluation of membrane protein assembly pathways in E. coli. In this study, we have analyzed the role of YidC in membrane integration of a widely used model IMP, leader peptidase (Lep). Site-directed photocross-linking experiments demonstrate that both YidC and SecY contact nascent Lep very early during biogenesis, at only 50-amino acid nascent chain length. At this length the first transmembrane domain (TM), which acquires a type I topology, is not even fully exposed outside the ribosome. The pattern of interactions appears dependent on the position of the cross-linking probe in the nascent chain. Upon elongation, nascent Lep remains close to YidC and comes into contact with lipids as well. Our results suggest a role for YidC in both the reception and lipid partitioning of type I TMs

A ban on BAM: an update on inhibitors of the β-barrel assembly machinery

Microbial Biotechnolog

Atypical periosteal osteoid osteoma: a case report

Osteoid osteoma is a benign osteoblastic tumor usually seen in adolescent and young males. In the paediatric age group, since the history may be difficult to elicit, there are often problems in early diagnosis. The author reports an unusual presentation of osteoid osteoma in a ten-year-old girl, which could not be diagnosed by conventional X-rays and CT scan

Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner membrane protein CyoA.

Inner membrane proteins (IMPs) of Escherichia coli use different pathways for membrane targeting and integration. YidC plays an essential but poorly defined role in the integration and folding of IMPs both in conjunction with the Sec translocon and as a Sec-independent insertase. Depletion of YidC only marginally affects the insertion of Sec-dependent IMPs, whereas it blocks the insertion of a subset of Sec-independent IMPs. Substrates of this latter "YidC-only" pathway include the relatively small IMPs M13 procoat, Pf3 coat protein, and subunit c of the

Synthesis and structure–activity studies of β-barrel assembly machine complex inhibitor MRL-494

Microbial Biotechnolog

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Evolocumab in pediatric heterozygous familial hypercholesterolemia

BACKGROUND Evolocumab, a fully human monoclonal antibody directed against proprotein convertase subtilisin–kexin type 9, is widely used in adult patients to lower low-density lipoprotein (LDL) cholesterol levels. Its effects in pediatric patients with heterozygous familial hypercholesterolemia are not known. METHODS We conducted a 24-week, randomized, double-blind, placebo-controlled trial to evaluate the efficacy and safety of evolocumab in pediatric patients with heterozygous familial hypercholesterolemia. Patients 10 to 17 years of age who had received stable lipid-lowering treatment for at least 4 weeks before screening and who had an LDL cholesterol level of 130 mg per deciliter (3.4 mmol per liter) or more and a triglyceride level of 400 mg per deciliter (4.5 mmol per liter) or less were randomly assigned in a 2:1 ratio to receive monthly subcutaneous injections of evolocumab (420 mg) or placebo. The primary end point was the percent change in LDL cholesterol level from baseline to week 24; key secondary end points were the mean percent change in LDL cholesterol level from baseline to weeks 22 and 24 and the absolute change in LDL cholesterol level from baseline to week 24. RESULTS A total of 157 patients underwent randomization and received evolocumab (104 patients) or placebo (53 patients). At week 24, the mean percent change from baseline in LDL cholesterol level was −44.5% in the evolocumab group and −6.2% in the placebo group, for a difference of −38.3 percentage points (P&lt;0.001). The absolute change in the LDL cholesterol level was −77.5 mg per deciliter (−2.0 mmol per liter) in the evolocumab group and −9.0 mg per deciliter (−0.2 mmol per liter) in the placebo group, for a difference of −68.6 mg per deciliter (−1.8 mmol per liter) (P&lt;0.001). Results for all secondary lipid variables were significantly better with evolocumab than with placebo. The incidence of adverse events that occurred during the treatment period was similar in the evolocumab and placebo groups. CONCLUSIONS In this trial involving pediatric patients with familial hypercholesterolemia, evolocumab reduced the LDL cholesterol level and other lipid variables