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Structure and Association of Human Lactoferrin Peptides with Escherichia coli Lipopolysaccharide. Daniel S. Chapple, 2004.An 11-amino-acid amphipathic synthetic peptide homologous to a helical region on helix 1 of human lactoferrin HLP-2 exhibited bactericidal activity against Escherichia coli serotype O111, whereas an analogue synthesized with Pro substituted for Met, HLP-6, had greatly reduced antimicrobial activity . The bactericidal activity of HLP-2 was 10-fold greater than that of HLP-6 in both buffer and growth medium by time-kill assays . These assays also showed a pronounced lag phase that was both concentration and time dependent and that was far greater for HLP-2 than for HLP-6 . Both peptides, however, were shown to be equally efficient in destabilizing the outer membrane when the hydrophobic probe 1-N-phenylnaphthylamine was used and to have the same lipopolysaccharide (LPS) binding affinity, as shown by polymyxin B displacement . Circular dichroism (CD) spectroscopy was used to study the structure and the organization of the peptides in solution and upon interaction with E . coli LPS . In the presence of LPS, HLP-2 and HLP-6 were found to bind and adopt a ß-strand conformation rather than an  -helix, as shown by nonimmobilized ligand interaction assay-CD spectroscopy . Furthermore, this assay was used to show that there is a time-dependent association of peptide that results in an ordered formation of peptide aggregates . The rate of interpeptide association was far greater in HLP-2 LPS than in HLP-6 LPS, which was consistent with the lag phase observed on the killing curves . These results allow us to propose a mechanism by which HLP-2 folds and self-assembles at the outer membrane surface before exerting its activity .
Regulation of the acuF Gene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous Fungus Aspergillus nidulans. Michael J. Hynes, 2002.Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle . Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK . The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi . The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters . Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate . Induction by acetate and proline is not additive, consistent with a single mechanism of induction . Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle . It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction . This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present . This difference in the control of gluconeogenesis reflects the ability of A . nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S . cerevisiae . The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S . cerevisiae Hap complex and the mammalian NFY complex . Global Regulation of the Salmonella enterica Serovar Typhimurium Major Porin, OmpD. Carlos A. Santiviago, 2003.The OmpD porin is the most abundant outer membrane protein in Salmonella enterica serovar Typhimurium and represents about 1% of total cell protein . Unlike the case with the less abundant OmpC and OmpF porins, the stoichiometry of OmpD in the outer membrane does not change in response to changes in osmolarity . The abundance of OmpD increases in response to anaerobiosis and decreases in response to low pH, conditions encountered by serovar Typhimurium during the infection of its murine host . By constructing an operon fusion of the lacZY genes with the ompD promoter, we show that the abundance of OmpD in the outer membrane is regulated primarily at the level of transcription and is subject to catabolite repression . In response to anaerobiosis, the abundance of OmpD in the outer membrane also appears to be controlled posttranscriptionally by a function dependent on Fnr . The Genes Coding for Enterocin EJ97 Production by Enterococcus faecalis EJ97 Are Located on a Conjugative Plasmid. Marina Sánchez-Hidalgo, 2003.Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da) . The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment . Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E . faecalis EJ97 to E . faecalis OG1X conferred bacteriocin production and resistance on the recipient . The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97 . This region was cloned and sequenced . It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC) . The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far . The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters . The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins . There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp) . ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216 . This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216 . ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E . faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon . Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response . These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E . faecalis, suggesting that they have a common origin .
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