CrrAB regulates PagP-mediated glycerophosphoglycerol palmitoylation in the outer membrane of Klebsiella pneumoniae

The outer membrane (OM) of Gram-negative bacteria is an evolving antibiotic barrier composed of a glycerophospholipid (GP) inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The two-component regulatory system CrrAB has only recently been reported to confer high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Mutations in crrB have been shown to lead to the modification of the lipid A moiety of LPS through CrrAB activation. However, functions of CrrAB activation in the regulation of other lipids are unclear. Work here demonstrates that CrrAB activation not only stimulates LPS modification but also regulates synthesis of acyl-glycerophosphoglycerols (acyl-PGs), a lipid species with undefined functions and biosynthesis. Among all possible modulators of acyl-PG identified from proteomic data, we found expression of lipid A palmitoyltransferase (PagP) was significantly upregulated in the crrB mutant. Furthermore, comparative lipidomics showed that most of the increasing acyl-PG activated by CrrAB was decreased after pagP knockout with CRISPR-Cas9. These results suggest that PagP also transfers a palmitate chain from GPs to PGs, generating acyl-PGs. Further investigation revealed that PagP mainly regulates the GP contents within the OM, leading to an increased ratio of acyl-PG to PG species and improving OM hydrophobicity, which may contribute to resistance against certain cationic antimicrobial peptides resistance upon LPS modification. Taken together, this work suggests that CrrAB regulates the palmitoylation of PGs and lipid A within the OM through upregulated PagP, which functions together to form an outer membrane barrier critical for bacterial survival.     

Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3'' processing signal AAUAAA.

Cleavage and polyadenylation factor (CPF) is required for the cleavage as well as for the subsequent polyadenylation reaction during 3' processing of messenger RNA precursors. Here, we have investigated the interaction of CPF and poly(A) polymerase with short RNA substrates. CPF activates poly(A) polymerase to elongate RNA primers carrying the canonical hexamer recognition signal AAUAAA. CPF specifically binds to such RNA as shown by gel mobility shift assays and competition experiments. Upon binding of CPF, two polypeptides of 35 kDa and 160 kDa can be covalently crosslinked to the RNA by irradiation with UV light. These polypeptides may correspond to the smallest and the largest subunit contained in purified CPF fractions. In addition, chemical modification-exclusion experiments demonstrate that CPF interacts directly with the AAUAAA recognition signal in the RNA. The entire hexamer signal is involved in binding of CPF since modification of any of its bases interferes with complex formation.     

Thymidine photoproducts of non-cyclobutane type as a crucial event in the conformation of UV-irradiated DNA

The UV-light induced conformational effects in deoxyoligonucleotides and polynucleotides have been analyzed by CD measurements and isolation of the photoproducts. The results demonstrate that the essential photoproduct formed on irradiation of thymidylyl-thymidine at 254 nm is of non-cyclobutane type and may be correlated to the primary photoproduct formed in DNAs at low doses Formation of thymine dimers of cyclobutane-type structure appears to be a secondary product generated by treatment with formic acid.     

An evaluation of analgesia associated with the immobility response in laboratory rabbits

The immobility response (IR) was studied in rabbits to evaluate its analgesic properties and reliability as a method of restraint. The participation of the endogenous opioid system in IR was studied indirectly by evaluating the effects of the narcotic antagonist naloxone on this phenomenon. Twenty-four adult New Zealand White rabbits were subjected to six noxious stimuli while restrained by IR and while restrained under control conditions. Testing on each animal was repeated under both conditions following the administration of naloxone. The noxious stimuli consisted of three levels of electric shock (10 volts, 30 volts, and 50 volts) applied to the shaved forearm, and mechanical pressure applied to the pinna, front toe, and hind toe. Withdrawal and changes in blood pressure, heart rate, and respiration were used as indicators of pain perception. Distress associated with noxious electrical and pressure stimulation was significantly reduced by IR, which suggested that the phenomenon does have a significant analgesic component. However, the rabbits showed wide variability in their susceptibility to IR induction, and even animals which did not withdraw in response to noxious stimulation under IR sometimes exhibited physiological changes suggestive of distress. Therefore, IR should not be considered as a reliable or humane alternative to analgesic/anesthetic drugs for laboratory rabbits. Naloxone had little effect on IR or IR-associated analgesia.     

Contrasting responses of oligochaete communities to the abatement of eutrophication in Lake Geneva

Tubificid and lumbriculid worms were used to monitor, at depths of 150 m, the recovery of Lake Geneva (Switzerland) from eutrophication. As predicted from the decrease of phosphorus concentrations, relative abundance of oligotrophic species was higher from 1988 to 1993 than in 1983, i.e. before the abatement of eutrophication. However, this trend towards oligotrophication can be reversed, as indicated by a decrease of oligotrophic species, recorded in 1993. But this change corresponded to the effects of an increase of water temperature on the abundance of the mesotrophic species Potamothrix vejdovskyi rather than to a deterioration of the profundal. In addition to this short-term setback, oligochaete communities located at a depth of 150 m responded more slowly and less clearly to the decrease of phosphorus concentrations than those located at a depth of 40 m. However, the zoobenthos indicated more clearly the recovery of Lake Geneva than the phytoplankton.