Biocides: mechanisms of action and microbial resistance

World Journal of Microbiology and Biotechnology -     

Bacillus cereus DNA topoisomerase I and IIIalpha: purification, characterization and complementation of Escherichia coli TopoIII activity

The Bacillus cereus genome possesses three type IA topoisomerase genes. These genes, encoding DNA topoisomerase I and IIIα (bcTopo I, bcTopo IIIα), have been cloned into T7 RNA polymerase-regulated plasmid expression vectors and the enzymes have been overexpressed, purified and characterized. The proteins exhibit similar biochemical activity to their Escherichia coli counterparts, DNA topoisomerase I and III (ecTopo I, ecTopo III). bcTopo I is capable of efficiently relaxing negatively supercoiled DNA in the presence of Mg²⁺ but does not possess an efficient DNA decatenation activity. bcTopo IIIα is an active topoisomerase that is capable of relaxing supercoiled DNA at a broad range of Mg²⁺ concentrations; however, its DNA relaxation activity is not as efficient as that of bcTopo I. In addition, bcTopo III is a potent DNA decatenase that resolves oriC-based plasmid replication intermediates in vitro. Interestingly, bcTopo I and bcTopo IIIα are both able to compensate for the loss of ecTopo III in E.coli cells that lack ecTopo I. In contrast, ecTopo I cannot substitute for ecTopo III under these conditions.     

Progress of structural genomics initiatives: an analysis of solved target structures

The explosion in gene sequence data and technological breakthroughs in protein structure determination inspired the launch of structural genomics (SG) initiatives. An often stated goal of structural genomics is the high-throughput structural characterisation of all protein sequence families, with the long-term hope of significantly impacting on the life sciences, biotechnology and drug discovery. Here, we present a comprehensive analysis of solved SG targets to assess progress of these initiatives. Eleven consortia have contributed 316 non-redundant entries and 323 protein chains to the Protein Data Bank (PDB), and 459 and 393 domains to the CATH and SCOP structure classifications, respectively. The quality and size of these proteins are comparable to those solved in traditional structural biology and, despite huge scope for duplicated efforts, only 14% of targets have a close homologue (>/=30% sequence identity) solved by another consortium. Analysis of CATH and SCOP revealed the significant contribution that structural genomics is making to the coverage of superfamilies and folds. A total of 67% of SG domains in CATH are unique, lacking an already characterised close homologue in the PDB, whereas only 21% of non-SG domains are unique. For 29% of domains, structure determination revealed a remote evolutionary relationship not apparent from sequence, and 19% and 11% contributed new superfamilies and folds. The secondary structure class, fold and superfamily distributions of this dataset reflect those of the genomes. The domains fall into 172 different folds and 259 superfamilies in CATH but the distribution is highly skewed. The most populous of these are those that recur most frequently in the genomes. Whilst 11% of superfamilies are bacteria-specific, most are common to all three superkingdoms of life and together the 316 PDB entries have provided new and reliable homology models for 9287 non-redundant gene sequences in 206 completely sequenced genomes. From the perspective of this analysis, it appears that structural genomics is on track to be a success, and it is hoped that this work will inform future directions of the field.     

Duration of alpha 1-antichymotrypsin gene activation by interleukin-1 is determined by efficiency of inhibitor of nuclear factor kappa B alpha resynthesis in primary human astrocytes

Expression of alpha1antichymotrypsin (ACT) is significantly activated by interleukin-1 (IL-1) in human astrocytes; however, it is barely affected by IL-1 in hepatocytes. This tissue-specific regulation depends upon an enhancer that contains both nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) elements, and is also observed for an NF-kappaB reporter but not for an AP-1 reporter. We found efficient activation of NF-kappaB binding in both cell types; however, this binding was persistent in glial cells and only transient in hepatocytes. IL-1-activated NF-kappaB complexes consisted of p65 and p50, with p65 transiently phosphorylated on serine 536 in glial cells whereas more persistently in hepatic cells. Overexpression of p65 or constitutively active IKKbeta (inhibitor of NF-kappaB kinase beta) resulted in an efficient activation of the ACT reporter in hepatic cells, indicating that a specific mechanism exists in these cells terminating IL-1 signaling. IL-1 effectively induced the degradation of inhibitor of NF-kappaBalpha (IkBalpha) and IkBepsilon in both cell types but IkBbeta was not affected. However, IkBalpha was resynthesized much more rapidly in hepatic cells in comparison to glial cells. In addition, the initial levels of IkBalpha were much lower in glial cells. We propose that the tissue-specific regulation of the ACT gene expression by IL-1 is determined by different efficiencies of IkBalpha resynthesis in glial and hepatic cells.     

Astrocyte- and hepatocyte-specific expression of genes from the distal serpin subcluster at 14q32.1 associates with tissue-specific chromatin structures

The distal serpin subcluster contains genes encoding alpha1-antichymotrypsin (ACT), protein C inhibitor (PCI), kallistatin (KAL) and the KAL-like protein, which are expressed in hepatocytes, but only the act gene is expressed in astrocytes. We show here that the tissue-specific expression of these genes associates with astrocyte- and hepatocyte-specific chromatin structures. In hepatocytes, we identified 12 Dnase I-hypersensitive sites (DHSs) that were distributed throughout the entire subcluster, with the promoters of expressed genes accessible to restriction enzyme digestion. In astrocytes, only six DHSs were located exclusively in the 5' flanking region of the act gene, with its promoter also accessible to restriction enzyme digestion. The acetylation of histone H3 and H4 was found throughout the subcluster in both cell types but this acetylation did not correlate with the expression pattern of these serpin genes. Analysis of histone modifications at the promoters of the act and pci genes revealed that methylation of histone H3 on lysine 4 correlated with their expression pattern in both cell types. In addition, inhibition of methyltransferase activity resulted in suppression of ACT and PCI mRNA expression. We propose that lysine 4 methylation of histone H3 correlates with the tissue-specific expression pattern of these serpin genes.     

Isolation of high molecular weight DNA suitable for BAC library construction from woody perennial soft-fruit species

We have developed a novel nuclei extraction method that allows for the extraction of high molecular weight DNA from leaves of woody perennial soft-fruit species that contain high levels of carbohydrates and polyphenolics. The method utilizes a modified buffer system including 4% (w/v) polyvinylpyrrolidone (PVP)-10 and a combination of nylon filters and Percoll gradients to purify nuclei extracts prior to embedding in agarose plugs. The effectiveness of the method was demonstrated on leaves of red raspberry (Rubus idaeus) and blackcurrant (Ribes nigrum), two soft-fruit species that have shown to be recalcitrant to standard genomic DNA extraction methods. Extracted DNA was readily digested by restriction enzymes and, as shown for raspberry, suitable for bacterial artificial chromosome (BAC) library construction.     

Doubling the cloak of (in)competence in client/therapist interactions

Cultural competence is used (often implicitly) to make decisions in human service settings. When therapists make decisions about whether or not a particular service will be offered, they place themselves in a position where their own competence can be judged. Using narrative data on independence and the elderly, we apply Edgerton's idea of the cloak of competence to demonstrate this doubling effect.     

Evolutionary relationships of three new species of Enterobacteriaceae living as symbionts of aphids and other insects

Ecological studies on three bacterial lineages symbiotic in aphids have shown that they impose a variety of effects on their hosts, including resistance to parasitoids and tolerance to heat stress. Phylogenetic analyses of partial sequences of gyrB and recA are consistent with previous analyses limited to 16S rRNA gene sequences and yield improved confidence of the evolutionary relationships of these symbionts. All three symbionts are in the Enterobacteriaceae. One of the symbionts, here given the provisional designation "Candidatus Serratia symbiotica," is a Serratia species that has acquired a symbiotic lifestyle. The other two symbionts, here designated "Candidatus Hamiltonella defensa" and "Candidatus Regiella insecticola," are sister groups to one another and together show a relationship to species of Photorhabdus.