Efficacy of a quaternary ammonium compound against planktonic and sessile populations of differentLegionella pneumophila strains

Efficacy of Gemacide PN-50^TM (a quaternary ammonium compound) as a commercial formulation recommended for disinfecting heat exchangers was determined for both planktonic and sessile populations of variousLegionella pneumophila strains. The quaternary ammonium compound (QAC) was preferred as an alternative due to the emerging resistance of potentially pathogenic bacteria against different biocides. PlanktonicL. pneumophila strains were suspended in tap water while sessile ones were grown on stainless steel that is used in construction of the cooling towers, then both group of strains were exposed to the biocide. The sensitivity of both planktonic and sessile populations ofL. pneumophila strains to the biocide was different. The biocide was found effective below recommended dosages (1000–2000 mg/L) against planktonic populations ofL. pneumophila, whereas it was determined that higher than the recommended dosages were required for sessile populations. The environmental isolates were more resistant to the biocide than the ATCC isolate was. The results indicated that studying only the planktonic populations ofL. pneumophila for biocide tests might not be sufficient to provide the optimum dosage and contact time information for field trials. Therefore, biocidal activity of a water treatment chemical must be evaluated in terms of dosage and contact times on both planktonic and sessile bacteria.     

Mitochondrial phylogeography of the long-eared bats (Plecotus) in the Mediterranean Palaearctic and Atlantic Islands

Long-eared bats of the genus Plecotus are widespread and common over most of the western Palaearctic. Based on recent molecular evidence, they proved to represent a complex of several cryptic species, with three new species being described from Europe in 2002. Evolutionary relationships among the different lineages are still fragmentary because of the limited geographic coverage of previous studies. Here we analyze Plecotus mitochondrial DNA sequences from the entire Mediterranean region and Atlantic Islands. Phylogenetic reconstructions group these western Palaearctic Plecotus into two major clades which split at least 5 Myr ago and that are each subdivided into further subgroups. An 'auritus group' includes the traditional P. auritus species and its sister taxon P. macrobullaris (=P. alpinus) plus related specimens from the Middle East. P. auritus and P. macrobullaris have broadly overlapping distributions in Europe, although the latter is apparently more restricted to mountain ranges. The other major clade, the 'austriacus group,' includes the European species P. austriacus and at least two other related taxa from North Africa (including P. teneriffae from the Canary Islands), the Balkans and Anatolia (P. kolombatovici). The sister species of this 'austriacus group' is P. balensis, an Ethiopian endemic. Phylogenetic reconstructions further suggest that P. austriacus reached Madeira during its relatively recent westward expansion through Europe, while the Canary Islands were colonized by a North African ancestor. Although colonization of the two groups of Atlantic Islands by Plecotus bats followed very distinct routes, neither involved lineages from the 'auritus group.' Furthermore, the Strait of Gibraltar perfectly segregates the distinct lineages, which confirms its key role as a geographic barrier. This study also stresses the biogeographical importance of the Mediterranean region, and particularly of North Africa, in understanding the evolution of the western Palaearctic biotas.     

Molecular ecology and phylogeography of the bent-wing bat complex (Miniopterus schreibersii) (Chiroptera: Vespertilionidae) in Asia Minor and adjacent regions

In this study we investigate population genetic structure and phylogeography of the bent-wing bat complex ( Miniopterus schreibersii ) in Asia Minor and adjacent regions. PCR amplification and sequencing of the first hypervariable domain of the mitochondrial control region were used to obtain the genetic data. Morphometric differentiation between lineages was analysed by comparing forearm lengths. We found two reciprocally monophyletic lineages within the M . schreibersii complex, identified as M.   s. schreibersii and M. s . pallidus . Distributions of the lineages were allopatric with a U-shaped suture zone passing through Central Anatolia. The suture zone separated coastal regions occupied by M.   s. schreibersii from inland, higher altitude regions occupied by M.   s. pallidus . The lineages showed a considerable sequence divergence of c . 9%, accompanied by a corresponding difference in forearm length. The presence of the genetically distinct lineages, with allopatric distribution and corresponding morphometric differences, probably reflects their long isolation during the ice-age in the Balkans and the Caspian/Caucasus refugia, followed subsequently by expansion into different habitats. Based on the present data, the lineages can be recognized as evolutionary significant units.     

Characterization of polVR391: a Y-family polymerase encoded by rumA'B from the IncJ conjugative transposon, R391

Although best characterized for their ability to traverse a variety of DNA lesions, Y-family DNA polymerases can also give rise to elevated spontaneous mutation rates if they are allowed to replicate undamaged DNA. One such enzyme that promotes high levels of spontaneous mutagenesis in Escherichia coli is polV(R391), a polV-like Y-family polymerase encoded by rumA'B from the IncJ conjugative transposon R391. When expressed in a DeltaumuDC lexA(Def) recA730 strain, polV(R391) promotes higher levels of spontaneous mutagenesis than the related MucA'B (polR1) or UmuD'C (polV) polymerases respectively. Analysis of the spectrum of polV(R391)-dependent mutations in rpoB revealed a unique genetic fingerprint that is typified by an increase in C:G-->A:T and A:T-->T:A transversions at certain mutagenic hot spots. Biochemical characterization of polV(R391) highlights the exceptional ability of the enzyme to misincorporate T opposite C and T in sequence contexts corresponding to mutagenic hot spots. Purified polV(R391) can also bypass a T-T pyrimidine dimer efficiently and displays greater accuracy opposite the 3'T of the dimer than opposite an undamaged T. Our study therefore provides evidence for the molecular basis for the enhanced spontaneous mutator activity of RumA'B, as well as explains its ability to promote efficient and accurate bypass of T-T pyrimidine dimers in vivo.     

Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli

The suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R-3-hydroxyacyl-ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42 degrees C. This was accompanied by a dramatic increase in the amount of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase [the IpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse-chase experiments and in vitro assays with purified components showed that FtsH, the AAA-type membrane-bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl-ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R-3-hydroxyacyl-ACP pool, is regulated by FtsH.     

Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin

Transposon mutagenesis was employed to isolate the gene(s) related with the biosynthesis of dipeptide antibiotic in Bacillus subtilis PY79 (a prototrophic derivative of the standard 168 strain). The blocked mutants were phenotypically selected from the transposon library by bioassay and the complete loss of biosynthetic ability was verified through ESI-mass spectrometry analysis. Four different bacilysin nonproducer mutants (Bac(-)::Tn10(ori-spc)) were isolated from the transposon library. The genes involved in bacilysin biosynthesis were identified as thyA (thymidilate synthetase), ybgG (unknown; similar to homocysteine methyl transferase) and oppA (oligopeptide permease), respectively. The other blocked gene was yvgW (unknown; similar to heavy metal-transporting ATPase); however, backcross studies did not verify its involvement in bacilysin biosynthesis. This gene, on the other hand, appeared to be necessary for efficient sporulation and transformation. Opp involvement was significant as it suggested that bacilysin biosynthesis is under or a component of the quorum sensing pathway which has been shown to be responsible for the establishment of sporulation, competence development and onset of surfactin biosynthesis. For verification, it was necessary to check the involvement of peptide pheromones (PhrA or PhrC) internalized by the Opp system and response regulator ComA as the essential components of this global control. phrA, phrC and comA deleted mutants of PY79 were thus constructed and the latter two genes were shown to be essential for bacilysin biosynthesis.     

Kinetic measurements of T----R structural transitions in insulin.

The T6----T3R3 and T3R3----R6-structural transitions of cobalt insulin hexamers as induced by SCN ions or m-cresol were studied in stopped-flow experiments using the absorption in the visible for monitoring their time course. The T6----T3R3 transition induced by either SCN or limited concentrations of m-cresol is mono-exponential with a rate constant of 0.1 s-1 and 0.4 s-1, respectively. A mono-exponential time course is also encountered for the m-cresol-induced T3R3----R6 transition when starting from the T3R3 state preestablished by either SCN or m-cresol. The corresponding rate constants are 1.3 s-1 and 0.49 s-1, respectively. If m-cresol is used beyond the concentration range where transformation is limited to one trimer, two exponentials are required for fitting the time course. The second exponential corresponds to the T3R3----R6 step with a concentration-independent rate constant of 0.4 s-1. The rate constant for the faster T6----T3R3 transition, however, increases with increasing excess of m-cresol.     

Diverse roles for histone H2A modifications in DNA damage response pathways in yeast

There are many types of DNA damage that are repaired by a multiplicity of different repair pathways. All damage and repair occur in the context of chromatin, and histone modifications are involved in many repair processes. We have analyzed the roles of H2A and its modifications in repair by mutagenizing modifiable residues in the N- and C-terminal tails of yeast H2A and by testing strains containing these mutations in multiple DNA repair assays. We show that residues in both tails are important for homologous recombination and nonhomologous end-joining pathways of double-strand break repair, as well as for survival of UV irradiation and oxidative damage. We show that H2A serine 122 is important for repair and/or survival in each of these assays. We also observe a complex pattern of H2A phosphorylation at residues S122, T126, and S129 in response to different damage conditions. We find that overlapping but nonidentical groups of H2A residues in both tails are involved in different pathways of repair. These data suggest the presence of a set of H2A "damage codes" in which distinct patterns of modifications on both tails of H2A may be used to identify specific types of damage or to promote specific repair pathways.