Absorption of pentachlorophenol (PCP) by bark chips and its role in microbial PCP degradation

A pentachlorophenol (PCP)-degrading mixed bacterial population was enriched in a biofilter filled with soft wood bark chips. We found that bark chips were essential for the degradation to proceed at PCP concentrations higher than 10M. PCP-degrading bacteria were found to be extremely sensitive to PCP. Bark chips absorbed PCP reversibly, thus detoxifying the medium and allowing degradation to proceed at higher concentrations of PCP (beyond 200M).     

A 4-bp deletion in the Birt-Hogg-Dubé gene (FLCN) causes dominantly inherited spontaneous pneumothorax

Primary spontaneous pneumothorax (PSP), a condition in which air enters the pleural space and causes secondary lung collapse, is mostly sporadic but also occurs in families. The precise etiology of PSP remains unknown, although it is associated with emphysemalike changes (bullae) in the lungs of almost all patients. We describe the results of a genetic study of a large Finnish family with a dominantly inherited tendency to PSP. A genomewide scan suggested linkage to chromosome 17p11. Screening of the best candidate gene, FLCN, revealed a 4-bp deletion in the first coding exon, which causes a frameshift that predicts a protein truncation 50 missense amino acids downstream. All carriers of the deletion had bullous lung lesions. Mutations in FLCN are also responsible for Birt-Hogg-Dubé (BHD) syndrome (a dominantly inherited disease characterized by benign skin tumors, PSP, and diverse types of renal cancer) and, rarely, are detected in sporadic renal and colorectal tumors. Unlike other FLCN mutations, the exon 4 deletion seems to be associated with bullous lung changes only with 100% penetrance. These results suggest that changes in FLCN may have an important role in the development of PSP and, more importantly, of emphysema, a chronic pulmonary disease that often leads to formation of bullous lesions and lowered pulmonary function. Additionally, given the strong association of PSP and BHD, the connection between these conditions needs to be investigated further, particularly in patients with familial PSP, who may be at a greater risk of developing renal cancer.     

Microbe repelling coated stainless steel analysed by field emission scanning electron microscopy and physicochemical methods

Coating of stainless steel with diamond-like carbon or certain fluoropolymers reduced or almost eliminated adhesion and biofilm growth of Staphylococcus epidermidis, Deinococcus geothermalis, Meiothermus silvanus and Pseudoxanthomonas taiwanensis. These species are known to be pertinent biofilm formers on medical implants or in the wet-end of paper machines. Field emission scanning electron microscopic analysis showed that Staph. epidermidis, D. geothermalis and M. silvanus grew on stainless steel using thread-like organelles for adhesion and biofilm formation. The adhesion threads were fewer in number on fluoropolymer-coated steel than on plain steel and absent when the same strains were grown in liquid culture. Psx. taiwanensis adhered to the same surfaces by a mechanism involving cell ghosts on which the biofilm of live cells grew. Hydrophilic (diamond-like carbon) or hydrophobic (fluoropolymer) coatings reduced the adherence of the four test bacteria on different steels. Selected topographic parameters, including root-mean-square roughness (S (q)), skewness (S (sk)) and surface kurtosis (S (ku)), were analysed by atomic force microscopy. The surfaces that best repelled microbial adhesion of the tested bacteria had higher skewness values than those only slightly repelling. Water contact angle, measured (theta (m)) or roughness corrected (theta (y)), affected the tendency for biofilm growth in a different manner for the four test bacteria.     

Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries

In the microarray format of the minisequencing method multiple oligonucleotide primers immobilised on a glass surface are extended with fluorescent ddNTPs using a DNA polymerase. The method is a promising tool for large-scale single nucleotide polymorphism (SNP) detection. We have compared eight chemical methods for covalent immobilisation of the oligonucleotide primers on glass surfaces. We included both commercially available, activated slides and slides that were modified by ourselves. In the comparison the differently derivatised glass slides were evaluated with respect to background fluorescence, efficiency of attaching oligonucleotides and performance of the primer arrays in minisequencing reactions. We found that there are significant differences in background fluorescence levels among the different coatings, and that the attachment efficiency, which was measured indirectly using extension by terminal transferase, varied largely depending on which immobilisation strategy was used. We also found that the attachment chemistry affects the genotyping accuracy, when minisequencing on microarrays is used as the genotyping method. The best genotyping results were observed using mercaptosilane-coated slides attaching disulfide-modified oligonucleotides.     

Bi- and trivalent glycopeptide mannopyranosides as inhibitors of type 1 fimbriae-mediated bacterial adhesion: variation of valency, aglycon and scaffolding

In order to test relevant structural parameters for effective inhibition of mannose-specific bacterial adhesion, bi- and trivalent glycopeptide α-d-mannopyranosides were synthesized that differ in their conformational properties as well as in the spatial arrangement of attached mannosyl residues. They were tested in an inhibition adhesion assay with fluorescent Escherichia coli bacteria and testing results were referenced to the inhibitory potency of methyl α-d-mannopyranoside. It was shown, that besides the nature of the mannoside aglycon moiety, scaffolding of α-d-mannopyranosides on a peptide backbone was important for the performance of the synthesized glycopeptides as inhibitors of bacterial adhesion.     

The Peptide Toxin Amylosin of Bacillus amyloliquefaciens from Moisture-Damaged Buildings Is Immunotoxic,Induces Potassium Efflux from Mammalian Cells,and Has Antimicrobial Activity

Amylosin, a heat-stable channel-forming non-ribosomally synthesized peptide toxin produced by strains of Bacillus amyloliquefaciens isolated from moisture-damaged buildings, is shown in this paper to have immunotoxic and cytotoxic effects on human cells as well as antagonistic effects on microbes. Human macrophages exposed to 50 ng of amylosin ml⁻¹ secreted high levels of cytokines interleukin-1β (IL-1β) and IL-18 within 2 h, indicating activation of the NLRP3 inflammasome, an integral part of the innate immune system. At the same exposure level, expression of IL-1β and IL-18 mRNA increased. Amylosin caused dose-dependent potassium ion efflux from all tested mammalian cells (human monocytes and keratinocytes and porcine sperm cells) at 1 to 2 μM exposure. Amylosin also inhibited the motility of porcine sperm cells and depolarized the mitochondria of human keratinocytes. Amylosin may thus trigger the activation of the NLRP3 inflammasome and subsequently cytokine release by causing potassium efflux from exposed cells. The results of this study indicate that exposure to amylosin activates the innate immune system, which could offer an explanation for the inflammatory symptoms experienced by occupants of moisture-damaged buildings. In addition, the amylosin-producing B. amyloliquefaciens inhibited the growth of both prokaryotic and eukaryotic indoor microbes, and purified amylosin also had an antimicrobial effect. These antimicrobial effects could make amylosin producers dominant and therefore significant causal agents of health problems in some moisture-damaged sites.     

ATGme: Open-source web application for rare codon identification and custom DNA sequence optimization

Background

Codon usage plays a crucial role when recombinant proteins are expressed in different organisms. This is especially the case if the codon usage frequency of the organism of origin and the target host organism differ significantly, for example when a human gene is expressed in E. coli. Therefore, to enable or enhance efficient gene expression it is of great importance to identify rare codons in any given DNA sequence and subsequently mutate these to codons which are more frequently used in the expression host.

Results

We describe an open-source web-based application, ATGme, which can in a first step identify rare and highly rare codons from most organisms, and secondly gives the user the possibility to optimize the sequence.

Conclusions

This application provides a simple user-friendly interface utilizing three optimization strategies: 1. one-click optimization, 2. bulk optimization (by codon-type), 3. individualized custom (codon-by-codon) optimization. ATGme is an open-source application which is freely available at: http://atgme.org     

Hydroxylation and Dechlorination of Tetrachlorohydroquinone by Rhodococcus sp. Strain CP-2 Cell Extracts

A cell extract of a polychlorophenol-degrading bacterium, Rhodococcus sp. strain CP-2, isolated from chlorophenol-contaminated soil, was shown to dechlorinate tetrachlorohydroquinone, the first intermediate in pentachlorophenol and 2,3,5,6-tetrachlorophenol degradation. Degradation of tetrachlorohydroquinone was catalyzed by a soluble enzyme(s). The reaction sequence for complete dechlorination involved hydroxylation and three reductive dechlorinations, producing 1,2,4-trihydroxybenzene. All chlorines were thus removed from the polychlorinated compound before ring cleavage.     

Penicillin-binding protein SpoVD disulphide is a target for StoA in Bacillus subtilis forespores

The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulphide bonds in hitherto unknown proteins in the intermembrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high-molecular-weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae , confirmed that a disulphide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD , stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulphide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.