Background:The mechanism of coordination between LPS synthesis and translocation is unknown. Results: Two new proteins, LapA and LapB, co-purify with LPS transport proteins. lapB mutants display defects in lipid A and core assembly. Conclusion: lapB mutants accumulate precursor LPS core species and exhibit elevated levels of LpxC. Significance: Coordinated assembly of LPS is a critical step for targeting to the outer membrane.

The RpoE factor is essential for the viability of Escherichia coli. RpoE regulates extracytoplasmic functions including lipopolysaccharide (LPS) translocation and some of its non-stoichiometric modifications. Transcription of the rpoE gene is positively autoregulated by E E and by unknown mechanisms that control the expression of its distally located promoter(s). Mapping of 5 ends of rpoE mRNA identified five new transcriptional initiation sites (P1 to P5) located distal to E E -regulated promoter. These promoters are activated in response to unique signals. Of these P2, P3, and P4 defined major promoters, recognized by RpoN, RpoD, and RpoS factors, respectively. Isolation of trans-acting factors, in vitro transcriptional and gel retardation assays revealed that the RpoN-recognized P2 promoter is positively regulated by a QseE/F two-component system and NtrC activator, whereas the RpoD-regulated P3 promoter is positively regulated by a Rcs system in response to defects in LPS core biosynthesis, overproduction of certain lipoproteins, and the global regulator CRP. Strains synthesizing Kdo 2 -LA LPS caused up to 7-fold increase in the rpoEP3 activity, which was abrogated in ⌬(waaC rcsB). Overexpression of a novel 73-nucleotide sRNA rirA (RfaH interacting RNA) generated by the processing of 5 UTR of the waaQ mRNA induces the rpoEP3 promoter activity concomitant with a decrease in LPS content and defects in the O-antigen incorporation. In the presence of RNA polymerase, RirA binds LPS regulator RfaH known to prevent premature transcriptional termination of waaQ and rfb operons. RirA in excess could titrate out RfaH causing LPS defects and the activation of rpoE transcription.The cell envelope of Gram-negative bacteria, including Escherichia coli, contains two distinct membranes, an inner (IM) 3 and an outer (OM) membrane separated by the periplasm, a hydrophilic compartment that includes a layer of peptidoglycan. The OM is an asymmetric lipid bilayer with phospholipids forming the inner leaflet and LPS forming the outer leaflet. LPSs are highly heterogeneous in composition. However, they share a common architecture composed of a membrane-anchored phosphorylated and acylated ␤(136)-linked GlcN disaccharide, termed lipid A, to which a carbohydrate moiety of varying size is attached (1, 2).During the analyses of signals that induce the RpoE-dependent extracytoplasmic stress response, we showed that strains synthesizing heptoseless LPS due to the absence of either GmhD (RfaD/HtrM) or WaaC exhibited a constitutive induction of RpoE (3-6). Such strains also display constitutive synthesis of exopolysaccharide that is regulated by the Rcs twocomponent system (5,7,8). Subsequently, we showed that strains synthesizing the minimal LPS structure composed of either Kdo 2 -lipid IV A or only free lipid IV A exhibited hyperelevated levels of the RpoE activity (6). The activity of RpoE is highly induced when the LPS assembly is severely compromised or when there is an imbalance between LPS and phospholipids (9). Lack of many LPS co...

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH2) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH2 resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the Candida genus (MICs in the 6.25–100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH2 was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH2 and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC50 values in the 10–100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH2 since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable.

Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl cis/trans isomerases (PPIs). The Escherichia coli cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed. Measurement of their PPIase activity revealed that PpiB is the major source of PPIase activity in the cytoplasm. Furthermore, viable Δ6ppi strains could be constructed only on minimal medium in the temperature range of 30–37 °C, but not on rich medium. To address the molecular basis of essentiality of PPIs, proteins that aggregate in their absence were identified. Next, wild-type and putative active site variants of FkpB, FklB, PpiB and PpiC were purified and in pull-down experiments substrates specific to each of these PPIs identified, revealing an overlap of some substrates. Substrates of PpiC were validated by immunoprecipitations using extracts from wild-type and PpiC-H81A strains carrying a 3xFLAG-tag appended to the C-terminal end of the ppiC gene on the chromosome. Using isothermal titration calorimetry, RpoE, RseA, S2, and AhpC were established as FkpB substrates and PpiC’s PPIase activity was shown to be required for interaction with AhpC.

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