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A Predicted ABC Transporter, FtsEX, Is Needed for Cell Division in Escherichia coli. Kari L. Schmidt, 2004.FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria . Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial . Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX . RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop . We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl . We also found that in wild-type E . coli both FtsE and FtsX localize to the division site . Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI . Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not . Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent . We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media . Biophysical Characterization of Endotoxin Inactivation by NK-2, an Antimicrobial Peptide Derived from Mammalian NK-Lysin. Jörg Andrä, 2004.NK-2, a membrane-acting antimicrobial peptide, was derived from the cationic core region of porcine NK-lysin and consists of 27 amino acid residues . It adopts an amphipathic,  -helical secondary structure and has been shown to interact specifically with membranes of negatively charged lipids . We therefore investigated the interaction of NK-2 with lipopolysaccharide (LPS), the main, highly anionic component of the outer leaflet of the outer membrane of gram-negative bacteria, by means of biophysical and biological assays . As model organisms and a source of LPS, we used Salmonella enterica strains with various lengths of the LPS carbohydrate moiety, including smooth LPS, rough LPS, and deep rough LPS (LPS Re) mutant strains . NK-2 binds to LPS Re with a high affinity and induces a change in the endotoxin-lipid A aggregate structure from a cubic or unilamellar structure to a multilamellar one . This structural change, in concert with a significant overcompensation of the negative charges of LPS, is thought to result in the neutralization of the endotoxic LPS activity in a cell culture system . Neutralization of LPS activity by NK-2 as well as its antibacterial activity against the various Salmonella strains strongly depends on the length of the sugar chains of LPS, with LPS Re being the most sensitive . This suggests that a hydrophobic peptide-LPS interaction is necessary for efficient neutralization of the biological activity of LPS and that the long carbohydrate chains, besides their function as a barrier for hydrophobic drugs, also serve as a trap for polycationic substances .
"Candidatus Hepatoplasma crinochetorum," a New, Stalk-Forming Lineage of Mollicutes Colonizing the Midgut Glands of a Terrestrial Isopod. Yongjie Wang, 2004.Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes . Phylogenetic analysis revealed that these symbionts represent a novel lineage of the Mollicutes and are only distantly related (<82% sequence identity) to members of the Mycoplasmatales and Entomoplasmatales . Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas . The same probe also detected morphotypically identical symbionts in other crinochete isopods . Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding . Based on the isolated phylogenetic position and unique cytological properties, the provisional name "Candidatus Hepatoplasma crinochetorum" is proposed for this new taxon of Mollicutes colonizing the hepatopancreas of P . scaber . Surveillance of Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Irish Liquid-Milk Pasteurization Plants To Determine the Incidence of Mycobacterium paratuberculosis. Ciara E. O'Reilly, 2004.Over the 13-month period from October 2000 to November 2001 (inclusive), the Food Safety Authority of Ireland (FSAI) carried out surveillance of Irish bulk raw (n = 389) and commercially pasteurized (n = 357) liquid-milk supplies to determine the incidence of Mycobacterium paratuberculosis . The pasteurization time-temperature conditions were recorded for all pasteurized samples . Overall, 56% of whole-milk pasteurized samples had been heat treated at or above a time-temperature combination of 75°C for 25 s . All analyses were undertaken at the Department of Food Science (Food Microbiology) laboratory at Queen's University Belfast . Each milk sample was subjected to two tests for M . paratuberculosis: immunomagnetic separation-PCR (IMS-PCR; to detect the presence of M . paratuberculosis cells, live or dead) and chemical decontamination and culture (to confirm the presence of viable M . paratuberculosis) . Overall, M . paratuberculosis DNA was detected by IMS-PCR in 50 (12.9%; 95% confidence interval, 9.9 to 16.5%) raw-milk samples and 35 (9.8%; 95% confidence interval, 7.1 to 13.3%) pasteurized-milk samples . Confirmed M . paratuberculosis was cultured from one raw-milk sample and no pasteurized-milk samples . It is concluded that M . paratuberculosis DNA is occasionally present at low levels in both raw and commercially pasteurized cows' milk . However, since no viable M . paratuberculosis was isolated from commercially pasteurized cows' milk on retail sale in the Republic of Ireland, current pasteurization procedures are considered to be effective . Genomic Variability of O Islands Encoding Tellurite Resistance in Enterohemorrhagic Escherichia coli O157:H7 Isolates. Diane E. Taylor, 2002.Strains of Escherichia coli causing enterohemorrhagic colitis belonging to the O157:H7 lineage are reported to be highly related . Fifteen strains of E . coli O157:H7 and 1 strain of E . coli O46:H- (nonflagellated) were examined for the presence of potassium tellurite resistance (Ter) . Ter genes comprising terABCDEF were shown previously to be part of a pathogenicity island also containing integrase, phage, and urease genes . PCR analysis, both conventional and light cycler based, demonstrated that about one-half of the Ter E . coli O157:H7 strains (6 of 15), including the Sakai strain, which has been sequenced, carried a single copy of the Ter genes . Five of the strains, including EDL933, which has also been sequenced, contained two copies . Three other O157:H7 strains and the O46:H- strain did not contain the Ter genes . In strains containing two copies, the Ter genes were associated with the serW and serX tRNA genes . Five O157:H7 strains resembled the O157 Sakai strain whose sequence contained one copy, close to serX, whereas in one isolate the single copy was associated with serW . There was no correlation between Ter and the ability to produce Shiga toxin ST1 or ST2 . The Ter MIC for most strains, containing either one or two copies, was 1,024 µg/ml, although for a few the MIC was intermediate, 64 to 128 µg/ml, which could be increased to 512 µg/ml by pregrowth of strains in subinhibitory concentrations of potassium tellurite . Reverse transcriptase PCR analysis confirmed that in most strains Ter was constitutive but that in the rest it was inducible and involved induction of terB and terC genes . Only the terB, -C, -D, and -E genes are required for Ter . The considerable degree of homology between the ter genes on IncH12 plasmid R478, which originated in Serratia marcescens, and pTE53, from an E . coli clinical isolate, suggests that the pathogenicity island was acquired from a plasmid . This work demonstrates diversity among E . coli O157:H7 isolates, at least as far as the presence of Ter genes is concerned . Genetic Analysis of the RcsC Sensor Kinase from Escherichia coli K-12. D. J. Clarke, 2002.The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli . RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase . Conversion of Lactobacillus pentosus D-Lactate Dehydrogenase to a D-Hydroxyisocaproate Dehydrogenase through a Single Amino Acid Replacement. Chizuka Tokuda, 2003.The single amino acid replacement of Tyr52 with Leu drastically increased the activity of Lactobacillus pentosus NAD-dependent D-lactate dehydrogenase toward larger aliphatic or aromatic 2-ketoacid substrates by 3 or 4 orders of magnitude and decreased the activity toward pyruvate by about 30-fold, converting the enzyme into a highly active D-2-hydroxyisocaproate dehydrogenase .
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