Sequence analysis of the plasmid pColG from the Escherichia coli strain CA46

The complete 4715 nucleotide sequence of the plasmid pColG from the Escherichia coli strain CA46, which was originally assumed to code for colicin G activity, has been determined. Based on the nucleotide sequence homology of the 1828bp replication region, with an average G+C content of 48%, pColG was classified as a ColE1-like plasmid. Computer assisted analysis of the remaining 2887bp nucleotide sequence with an average G+C content of 34% revealed three putative OFRs. To find out whether one or all of the three ORFs code for a possible bacteriocin, a DNA fragment encompassing these ORFs has been cloned and the recombinant colonies tested for bacteriocin production. None of the colonies had an inhibitory activity against E. coli strains DH5, HB101 and MC4100. The assumption that the plasmid pColG from the E. coli strain CA46 codes for a bacteriocin thus could not be confirmed.     

Diversity of halophilic archaea in the crystallizers of an Adriatic solar saltern

Haloarchaeal diversity in the crystallizers of Adriatic Secovlje salterns was investigated using gene fragments encoding 16S rRNA and bacteriorhodopsin as molecular markers. Screening of 180 clones from five gene libraries constructed for each gene targeted revealed 15 different 16S rRNA and 10 different bacteriorhodopsin phylotypes, indicating higher haloarchaeal diversity than previously reported in such hypersaline environments. Furthermore, results of rarefaction analysis indicated that analysis of an increasing number of clones would have revealed additional diversity. Finally, most sequences from the crystallizers grouped within the Halorubrum branch, whereas square-shaped 'Haloquadratum' relatives, repeatedly reported to dominate crystallizer communities, were rare. Presence of such special and diverse haloarchaeal community could be attributed to the Secovlje salterns rare continuous short-cycling salt production mechanism.     

Impact of Carrier Systems on the Interactions of Coenzyme Q<Subscript>10</Subscript> with Model Lipid Membranes

We investigated the influence of carrier systems for different commercially available water-soluble formulations for coenzyme Q₁₀ on structural changes of model lipid membranes formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and by a mixture of phosphatidylcholine and sphingomyelin (2.4:1). Structural changes in the membranes were measured using fluorescence anisotropy, electron paramagnetic resonance, and differential scanning calorimetry. Two fluorophores and two spin probes were used to monitor membrane characteristics close to the water-lipid interface and in the middle of the bilayer of the model lipid membranes. Different water-soluble carrier systems were tested. These data show that different systems can facilitate penetration of CoQ₁₀ in the lipid membranes, where an increase in the lipid order parameter was observed. In addition, water soluble CoQ₁₀ formulations better protect lipids from oxidation in liposome solution. With the exception of the carriers in an emulsified formulation of CoQ₁₀, those in the other samples did not have any significant effects on membrane fluidity.     

Regulation of the major proline permease gene of Salmonella typhimurium.

The structural gene for the major proline permease is located in a tight cluster with genes coding for the proline degradative enzymes, proline oxidase and pyrroline-5-carboxylic acid dehydrogenase. Expression of the permease is regulated in parallel with the two degradative enzymes, and all three functions are subject to catabolite repression. Regulatory mutants (putC) have constitutively high levels of all three activities, suggesting that all are regulated by a single mechanism.     

Oral bioavailability of silymarin phytocomplex formulated as self-emulsifying pellets

The objective of this study was to develop new solid self-emulsifying pellets to deliver milk thistle extract (silymarin). These pellets were prepared via extrusion/spheronisation procedure, using a self-emulsifying system or SES (Akoline MCM^®, Miglyol^®, Tween 80^®, soy lecithin and propylene glycol), microcrystalline cellulose and lactose monohydrate. To select the most suitable formulations for extrusion and spheronisation, an experimental design of experiences was adopted. The screening amongst formulations (13 different blends) was performed preparing pellets and evaluating extrusion profiles and quality of the spheronised extrudates. The pellets were characterised for size and shape, density, force required to crush them. Although more than one type of pellets demonstrated adequate morphological and technological characteristics, pellets prepared from formulation 7 revealed the best properties and were selected for further biopharmaceutical investigations, including in vitro dissolution and in vivo trials on rats to study serum and lymph levels after oral administration of the pellets. These preliminary technological and pharmacokinetic data demonstrated that extrusion/spheronisation is a viable technology to produce self-emulsifying pellets of good quality and able to improve in vivo oral bioavailability of main components of a phytotherapeutic extract of more than 100 times by enhancing the lymphatic route of absorption.     

Haloarchaeal communities in the crystallizers of two adriatic solar salterns

Solar salterns operate only for short dry periods of the year in the north shore of the Adriatic Sea because of its relatively humid and cold Mediterranean climate. In a previous paper, we showed that the NaCl precipitation ponds (crystallizers) of Northern Adriatic Secovlje salterns have different haloarchaeal populations from those typically found in dry and hot climates such as Southern Spain. To check whether there is a common pattern of haloarchaeal diversity in these less extreme conditions, diversity in crystallizers of other Adriatic solar salterns in Ston, Croatia was ascertained by molecular and culture methods. In addition, the cultivation approach was used to further describe haloarchaeal diversity in both salterns. Over the period of two solar salt collection seasons, isolates related to species of the genera Haloferax, Haloarcula, and Haloterrigena were recovered from both salterns. Within the same sampling effort, relatives of the genus Halorubrum and a Natrinema-like isolate were cultivated from Slovenian Secovlje salterns while Halobacterium related isolates were obtained from the Croatian Ston salterns. Concurrent with our previous findings, a library of Croatian saltern crystallizer PCR-amplified 16S rRNA genes was dominated by sequences related to the genus Halorubrum. The microbial community structure was similar in both salterns but diversity indices showed greater values in Slovenian salterns when compared with Croatian salterns.     

Aerobactin uptake system, ColV production, and drug resistance encoded by a plasmid from an urinary tract infection Escherichia coli strain of human origin

A study of Escherichia coli strains isolated from patients suffering from urinary tract infections in Ljubljana, Yugoslavia, revealed a plasmid encoding the aerobactin iron uptake system, ColV production, and drug resistance. The plasmid is conjugative and at least 85 kilobases in length.     

A nonsense mutation abrogates production of a functional enterotoxin A in Clostridium difficile toxinotype VIII strains of serogroups F and X

Clostridium difficile strains of toxinotype VIII from serogroups F and X are described as toxin B-positive, toxin A-negative (TcdB+ A-), although they harbour almost the entire tcdA gene. To identify the reason for the lack of TcdA detection, we analyzed catalytic and ligand domains of TcdA-1470 of the type strain of serogroup F, strain 1470. Using recombinant fragments, the C-terminal immunodominant ligand domain TcdA3-1470, spanning amino acid residues 1694-2711 (corresponding to VPI 10463 sequence), was detected in Western blots. Similar experiments using the recombinant N-terminal catalytic fragment TcdAc1-2-1470 (amino acid positions 1-544) failed. In addition, this fragment showed no glucosylation activity. We determined the size and the position of alterations in the ligand domain tcdA3-1470 by DNA sequencing. Within the N-terminal fragment tcdAc1-2-1470, a nonsense mutation was identified introducing a stop codon at amino acid position 47. Identical mutations were found in the two serogroup X strains 17663 and 10355. The mutation might explain the lack of TcdA production observed in strains of serotypes F and X.     

Binary toxin producing Clostridium difficile strains

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.     

Sporulation of a bacitracin-sensitive mutant of Bacillus licheniformis is self-inhibited by bacitracin

A mutant of Bacillus licheniformis (BLU166) sensitive to its own antibiotic bacitracin was isolated and the mutation bcr-l was mapped close to the bacitracin synthetase genes. The sensitivity was shown to be specific for bacitracin. Two further bacitracin-sensitive strains were constructed, one (BLU171) with normal ability to synthesize bacitracin, and one (BLU170) a bacitracin non-producer. In addition to an increased sensitivity of growing cells to bacitracin, sporulation of the mutant strain BLU171 was self-inhibited by bacitracin. It is concluded that (1) there might exist at least two levels of resistance to bacitracin; (2) mutation bcr-1 affects a 'structural' component, which may protect the sensitive reaction of cell-wall biosynthesis; (3) sporulation is affected to a greater extent by bacitracin than vegetative growth; and (4) synthesis of bacitracin is independent of the presence of this resistance mechanism since the sensitive mutant produces similar amounts of the antibiotic to the wild-type strain.