Novel whole-cell antibiotic biosensors for compound discovery

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of 相似文献   

MALDI-TOF MS of phosphatidylethanolamines: different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described. In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

Methane and sulfate profiles within the subsurface of a tidal flat are reflected by the distribution of sulfate-reducing bacteria and methanogenic archaea

The anoxic layers of marine sediments are dominated by sulfate reduction and methanogenesis as the main terminal oxidation processes. The aim of this study was to analyze the vertical succession of microbial populations involved in these processes along the first 4.5 m of a tidal-flat sediment. Therefore, a quantitative PCR approach was applied using primers targeting the domains of Bacteria and Archaea, and key functional genes for sulfate reduction (dsrA) and methanogenesis (mcrA). The sampling site was characterized by an unusual sulfate peak at 250 cm depth resulting in separate sulfate-methane transition zones. Methane and sulfate profiles were diametrically opposed, with a methane maximum in the sulfate-depleted zone showing high numbers of archaea and methanogens. The methane-sulfate interfaces harbored elevated numbers of sulfate reducers, and revealed a slight increase in mcrA and archaeal 16S rRNA genes, suggesting sulfate-dependent anaerobic oxidation of methane. A diversity analysis of both functional genes by PCR-denaturing gradient gel electrophoresis revealed a vertical succession of subpopulations that were governed by geochemical and sedimentologic conditions. Along the upper 200 cm, sulfate-reducing populations appeared quite uniform and were dominated by the Deltaproteobacteria. In the layers beneath, an apparent increase in diversity and a shift to the Firmicutes as the predominant group was observed.     

The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter

The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [³H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.     

Tropheryma whipplei in the Environment: Survey of Sewage Plant Influxes and Sewage Plant Workers

We studied the prevalence of Tropheryma whipplei in influxes to 46 sewage treatment plants and in stool, mouthwash fluids, and dental plaques of 64 healthy workers in those facilities and 146 disease control patients. T. whipplei was found in sewage water, in stool of healthy individuals, and significantly more often in stool of workers exposed to sewage water.     

Functional interactions between Prp8, Prp18, Slu7, and U5 snRNA during the second step of pre-mRNA splicing

After the second transesterification step of pre-mRNA splicing, the Prp22 helicase catalyzes release of spliced mRNA by disrupting contacts in the spliceosome that likely involve Prp8. Mutations at Arg1753 in Prp8, which suppress helicase-defective prp22 mutants, elicit temperature-sensitive growth phenotypes, indicating that interactions in the spliceosome involving Prp8-R1753 might be broken prematurely at 37 degrees C. Here we report that mutations in loop I of the U5 snRNA or in Prp18 can suppress the temperature-sensitive prp8-R1753 mutants. The same gain-of-function PRP18 alleles can also alleviate the growth phenotypes of multiple slu7-ts mutants, indicating a functional link between Prp8 and the second step splicing factors Prp18 and Slu7. These findings, together with the demonstration that changes at Arg1753 in Prp8 impair step 2 of pre-mRNA splicing in vitro, are consistent with a model in which (1) Arg1753 plays a role in stabilizing U5/exon interactions prior to exon joining and (2) these contacts persist until they are broken by the helicase Prp22.