Food systems microbiome-related educational needs.

Within the European-funded Coordination and Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), the Workshop 'Education in Food Systems Microbiome Related Sciences: Needs for Universities, Industry and Public Health Systems' brought together over 70 researchers, public health and industry partners from all over the world to work on elaborating microbiome-related educational needs in food systems. This publication provides a summary of discussions held during and after the workshop and the resulting recommendations.

Targeted Metabolomics and High-Throughput RNA Sequencing-Based Transcriptomics Reveal Massive Changes in the Streptomyces venezuelae NRRL B-65442 Metabolism Caused by Ethanol Shock.

The species Streptomyces venezuelae is represented by several distinct strains with variable abilities to biosynthesize structurally diverse secondary metabolites. In this work, we examined the effect of ethanol shock on the transcriptome and metabolome of Streptomyces venezuelae NRRL B-65442 using high-throughput RNA sequencing (RNA-seq) and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ethanol shock caused massive changes in the gene expression profile, differentially affecting genes for secondary metabolite biosynthesis and central metabolic pathways. Most of the data from the transcriptome analysis correlated well with the metabolome changes, including the overproduction of jadomycin congeners and a downshift in the production of desferrioxamines, legonoxamine, foroxymithin, and a small cryptic ribosomally synthesized peptide. Some of the metabolome changes, such as the overproduction of chloramphenicol, could not be explained by overexpression of the cognate biosynthetic genes but correlated with the expression profiles of genes for precursor biosynthesis. Changes in the transcriptome were also observed for several genes known to play a role in stress response in other bacteria and included at least 10 extracytoplasmic function σ factors. This study provides important new insights into the stress response in antibiotic-producing bacteria and will help to understand the complex mechanisms behind the environmental factor-induced regulation of secondary metabolite biosynthesis. IMPORTANCE Streptomyces spp. are filamentous Gram-positive bacteria known as versatile producers of secondary metabolites, of which some have been developed into human medicines against infections and cancer. The genomes of these bacteria harbor dozens of gene clusters governing the biosynthesis of secondary metabolites (BGCs), of which most are not expressed under laboratory conditions. Detailed knowledge of the complex regulation of BGC expression is still lacking, although certain growth conditions are known to trigger the production of previously undetected secondary metabolites. In this work, we investigated the effect of ethanol shock on the production of secondary metabolites by Streptomyces venezuelae and correlated these findings with the expression of cognate BGCs and primary metabolic pathways involved in the generation of cofactors and precursors. The findings of this study set the stage for the rational manipulation of bacterial genomes aimed at enhanced production of industrially important bioactive natural products.

A time-resolved multi-omics atlas of Acanthamoeba castellanii encystment.

Encystment is a common stress response of most protists, including free-living amoebae. Cyst formation protects the amoebae from eradication and can increase virulence of the bacteria they harbor. Here, we mapped the global molecular changes that occur in the facultatively pathogenic amoeba Acanthamoeba castellanii during the early steps of the poorly understood process of encystment. By performing transcriptomic, proteomic, and phosphoproteomic experiments during encystment, we identified more than 150,000 previously undescribed transcripts and thousands of protein sequences absent from the reference genome. These results provide molecular details to the regulation of expected biological processes, such as cell proliferation shutdown, and reveal new insights such as a rapid phospho-regulation of sites involved in cytoskeleton remodeling and translation regulation. This work constitutes the first time-resolved molecular atlas of an encysting organism and a useful resource for further investigation of amoebae encystment to allow for a better control of pathogenic amoebae.

Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized gut microbiome until the Baroque period.

We subjected human paleofeces dating from the Bronze Age to the Baroque period (18th century AD) to in-depth microscopic, metagenomic, and proteomic analyses. The paleofeces were preserved in the underground salt mines of the UNESCO World Heritage site of Hallstatt in Austria. This allowed us to reconstruct the diet of the former population and gain insights into their ancient gut microbiome composition. Our dietary survey identified bran and glumes of different cereals as some of the most prevalent plant fragments. This highly fibrous, carbohydrate-rich diet was supplemented with proteins from broad beans and occasionally with fruits, nuts, or animal food products. Due to these traditional dietary habits, all ancient miners up to the Baroque period have gut microbiome structures akin to modern non-Westernized individuals whose diets are also mainly composed of unprocessed foods and fresh fruits and vegetables. This may indicate a shift in the gut community composition of modern Westernized populations due to quite recent dietary and lifestyle changes. When we extended our microbial survey to fungi present in the paleofeces, in one of the Iron Age samples, we observed a high abundance of Penicillium roqueforti and Saccharomyces cerevisiae DNA. Genome-wide analysis indicates that both fungi were involved in food fermentation and provides the first molecular evidence for blue cheese and beer consumption in Iron Age Europe.

Great Cause-Small Effect: Undeclared Genetically Engineered Orange Petunias Harbor an Inefficient Dihydroflavonol 4-Reductase.

A recall campaign for commercial, orange flowering petunia varieties in spring 2017 caused economic losses worldwide. The orange varieties were identified as undeclared genetically engineered (GE)-plants, harboring a maize dihydroflavonol 4-reductase (DFR, A1), which was used in former scientific transgenic breeding attempts to enable formation of orange pelargonidin derivatives from the precursor dihydrokaempferol (DHK) in petunia. How and when the A1 cDNA entered the commercial breeding process is unclear. We provide an in-depth analysis of three orange petunia varieties, released by breeders from three countries, with respect to their transgenic construct, transcriptomes, anthocyanin composition, and flavonoid metabolism at the level of selected enzymes and genes. The two possible sources of the A1 cDNA in the undeclared GE-petunia can be discriminated by PCR. A special version of the A1 gene, the A1 type 2 allele, is present, which includes, at the 3'-end, an additional 144 bp segment from the non-viral transposable Cin4-1 sequence, which does not add any functional advantage with respect to DFR activity. This unequivocally points at the first scientific GE-petunia from the 1980s as the A1 source, which is further underpinned e.g., by the presence of specific restriction sites, parts of the untranslated sequences, and the same arrangement of the building blocks of the transformation plasmid used. Surprisingly, however, the GE-petunia cannot be distinguished from native red and blue varieties by their ability to convert DHK in common in vitro enzyme assays, as DHK is an inadequate substrate for both the petunia and maize DFR. Recombinant maize DFR underpins the low DHK acceptance, and, thus, the strikingly limited suitability of the A1 protein for a transgenic approach for breeding pelargonidin-based flower color. The effect of single amino acid mutations on the substrate specificity of DFRs is demonstrated. Expression of the A1 gene is generally lower than the petunia DFR expression despite being under the control of the strong, constitutive p35S promoter. We show that a rare constellation in flavonoid metabolism-absence or strongly reduced activity of both flavonol synthase and B-ring hydroxylating enzymes-allows pelargonidin formation in the presence of DFRs with poor DHK acceptance.

Growth of Chlamydia pneumoniae Is Enhanced in Cells with Impaired Mitochondrial Function.

Effective growth and replication of obligate intracellular pathogens depend on host cell metabolism. How this is connected to host cell mitochondrial function has not been studied so far. Recent studies suggest that growth of intracellular bacteria such as Chlamydia pneumoniae is enhanced in a low oxygen environment, arguing for a particular mechanistic role of the mitochondrial respiration in controlling intracellular progeny. Metabolic changes in C. pneumoniae infected epithelial cells were analyzed under normoxic (O2 ≈ 20%) and hypoxic conditions (O2 < 3%). We observed that infection of epithelial cells with C. pneumoniae under normoxia impaired mitochondrial function characterized by an enhanced mitochondrial membrane potential and ROS generation. Knockdown and mutation of the host cell ATP synthase resulted in an increased chlamydial replication already under normoxic conditions. As expected, mitochondrial hyperpolarization was observed in non-infected control cells cultured under hypoxic conditions, which was beneficial for C. pneumoniae growth. Taken together, functional and genetically encoded mitochondrial dysfunction strongly promotes intracellular growth of C. pneumoniae.

Comprehensive Identification of Meningococcal Genes and Small Noncoding RNAs Required for Host Cell Colonization.

UNLABELLED: Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N. meningitidis life cycle that is poorly understood. Here, highly saturated random transposon insertion libraries of N. meningitidis were engineered, and the fitness of mutations during routine growth and that of colonization of endothelial and epithelial cells in a flow device were assessed in a transposon insertion site sequencing (Tn-seq) analysis. This allowed the identification of genes essential for bacterial growth and genes specifically required for host cell colonization. In addition, after having identified the small noncoding RNAs (sRNAs) located in intergenic regions, the phenotypes associated with mutations in those sRNAs were defined. A total of 383 genes and 8 intergenic regions containing sRNA candidates were identified to be essential for growth, while 288 genes and 33 intergenic regions containing sRNA candidates were found to be specifically required for host cell colonization. IMPORTANCE: Meningococcal meningitis is a common cause of meningitis in infants and adults. Neisseria meningitidis (meningococcus) is also a commensal bacterium of the nasopharynx and is carried by 3 to 30% of healthy humans. Under some unknown circumstances, N. meningitidis is able to invade the bloodstream and cause either meningitis or a fatal septicemia known as purpura fulminans. The onset of symptoms is sudden, and death can follow within hours. Although many meningococcal virulence factors have been identified, the mechanisms that allow the bacterium to switch from the commensal to pathogen state remain unknown. Therefore, we used a Tn-seq strategy coupled to high-throughput DNA sequencing technologies to find genes for proteins used by N. meningitidis to specifically colonize epithelial cells and primary brain endothelial cells. We identified 383 genes and 8 intergenic regions containing sRNAs essential for growth and 288 genes and 33 intergenic regions containing sRNAs required specifically for host cell colonization.

Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.

UNLABELLED: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.

Metagenomic analysis reveals presence of Treponema denticola in a tissue biopsy of the Iceman.

Ancient hominoid genome studies can be regarded by definition as metagenomic analyses since they represent a mixture of both hominoid and microbial sequences in an environment. Here, we report the molecular detection of the oral spirochete Treponema denticola in ancient human tissue biopsies of the Iceman, a 5,300-year-old Copper Age natural ice mummy. Initially, the metagenomic data of the Iceman's genomic survey was screened for bacterial ribosomal RNA (rRNA) specific reads. Through ranking the reads by abundance a relatively high number of rRNA reads most similar to T. denticola was detected. Mapping of the metagenome sequences against the T. denticola genome revealed additional reads most similar to this opportunistic pathogen. The DNA damage pattern of specifically mapped reads suggests an ancient origin of these sequences. The haematogenous spread of bacteria of the oral microbiome often reported in the recent literature could already explain the presence of metagenomic reads specific for T. denticola in the Iceman's bone biopsy. We extended, however, our survey to an Iceman gingival tissue sample and a mouth swab sample and could thereby detect T. denticola and Porphyrimonas gingivalis, another important member of the human commensal oral microflora. Taken together, this study clearly underlines the opportunity to detect disease-associated microorganisms when applying metagenomics-enabled approaches on datasets of ancient human remains.

Ultra deep sequencing of Listeria monocytogenes sRNA transcriptome revealed new antisense RNAs.

Listeria monocytogenes, a gram-positive pathogen, and causative agent of listeriosis, has become a widely used model organism for intracellular infections. Recent studies have identified small non-coding RNAs (sRNAs) as important factors for regulating gene expression and pathogenicity of L. monocytogenes. Increased speed and reduced costs of high throughput sequencing (HTS) techniques have made RNA sequencing (RNA-Seq) the state-of-the-art method to study bacterial transcriptomes. We created a large transcriptome dataset of L. monocytogenes containing a total of 21 million reads, using the SOLiD sequencing technology. The dataset contained cDNA sequences generated from L. monocytogenes RNA collected under intracellular and extracellular condition and additionally was size fractioned into three different size ranges from <40 nt, 40-150 nt and >150 nt. We report here, the identification of nine new sRNAs candidates of L. monocytogenes and a reevaluation of known sRNAs of L. monocytogenes EGD-e. Automatic comparison to known sRNAs revealed a high recovery rate of 55%, which was increased to 90% by manual revision of the data. Moreover, thorough classification of known sRNAs shed further light on their possible biological functions. Interestingly among the newly identified sRNA candidates are antisense RNAs (asRNAs) associated to the housekeeping genes purA, fumC and pgi and potentially their regulation, emphasizing the significance of sRNAs for metabolic adaptation in L. monocytogenes.

MScDB: a mass spectrometry-centric protein sequence database for proteomics.

Protein sequence databases are indispensable tools for life science research including mass spectrometry (MS)-based proteomics. In current database construction processes, sequence similarity clustering is used to reduce redundancies in the source data. Albeit powerful, it ignores the peptide-centric nature of proteomic data and the fact that MS is able to distinguish similar sequences. Therefore, we introduce an approach that structures the protein sequence space at the peptide level using theoretical and empirical information from large-scale proteomic data to generate a mass spectrometry-centric protein sequence database (MScDB). The core modules of MScDB are an in-silico proteolytic digest and a peptide-centric clustering algorithm that groups protein sequences that are indistinguishable by mass spectrometry. Analysis of various MScDB uses cases against five complex human proteomes, resulting in 69 peptide identifications not present in UniProtKB as well as 79 putative single amino acid polymorphisms. MScDB retains ~99% of the identifications in comparison to common databases despite a 3-48% increase in the theoretical peptide search space (but comparable protein sequence space). In addition, MScDB enables cross-species applications such as human/mouse graft models, and our results suggest that the uncertainty in protein assignments to one species can be smaller than 20%.

MIPS: curated databases and comprehensive secondary data resources in 2010.

The Munich Information Center for Protein Sequences (MIPS at the Helmholtz Center for Environmental Health, Neuherberg, Germany) has many years of experience in providing annotated collections of biological data. Selected data sets of high relevance, such as model genomes, are subjected to careful manual curation, while the bulk of high-throughput data is annotated by automatic means. High-quality reference resources developed in the past and still actively maintained include Saccharomyces cerevisiae, Neurospora crassa and Arabidopsis thaliana genome databases as well as several protein interaction data sets (MPACT, MPPI and CORUM). More recent projects are PhenomiR, the database on microRNA-related phenotypes, and MIPS PlantsDB for integrative and comparative plant genome research. The interlinked resources SIMAP and PEDANT provide homology relationships as well as up-to-date and consistent annotation for 38,000,000 protein sequences. PPLIPS and CCancer are versatile tools for proteomics and functional genomics interfacing to a database of compilations from gene lists extracted from literature. A novel literature-mining tool, EXCERBT, gives access to structured information on classified relations between genes, proteins, phenotypes and diseases extracted from Medline abstracts by semantic analysis. All databases described here, as well as the detailed descriptions of our projects can be accessed through the MIPS WWW server (http://mips.helmholtz-muenchen.de).

Impact of natural genetic variation on the transcriptome of autotetraploid Arabidopsis thaliana.

Polyploidy, the presence of more than two complete sets of chromosomes in an organism, has significantly shaped the genomes of angiosperms during evolution. Two forms of polyploidy are often considered: allopolyploidy, which originates from interspecies hybrids, and autopolyploidy, which originates from intraspecies genome duplication events. Besides affecting genome organization, polyploidy generates other genetic effects. Synthetic allopolyploid plants exhibit considerable transcriptome alterations, part of which are likely caused by the reunion of previously diverged regulatory hierarchies. In contrast, autopolyploids have relatively uniform genomes, suggesting lower alteration of gene expression. To evaluate the impact of intraspecies genome duplication on the transcriptome, we generated a series of unique Arabidopsis thaliana autotetraploids by using different ecotypes. A. thaliana autotetraploids show transcriptome alterations that strongly depend on their parental genome composition and include changed expression of both new genes and gene groups previously described from allopolyploid Arabidopsis. Alterations in gene expression are stable, nonstochastic, developmentally specific, and associated with changes in DNA methylation. We propose that Arabidopsis possesses an inherent and heritable ability to sense and respond to elevated, yet balanced chromosome numbers. The impact of natural variation on alteration of autotetraploid gene expression stresses its potential importance in the evolution and breeding of plants.

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron-reducing enrichment culture.

Anaerobic benzene degradation was studied with a highly enriched iron-reducing culture (BF) composed of mainly Peptococcaceae-related Gram-positive microorganisms. The proteomes of benzene-, phenol- and benzoate-grown cells of culture BF were compared by SDS-PAGE. A specific benzene-expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N-terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI-MS/MS-based shotgun proteomic analysis revealed other specifically benzene-expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate-CoA ligase (BamY) of Geobacter metallireducens and 67% to 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (∼ 17 kb) composed of carboxylase-related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX).

Independent evolution of the core domain and its flanking sequences in small heat shock proteins.

Small heat shock proteins (sHsps) are molecular chaperones involved in maintaining protein homeostasis; they have also been implicated in protein folding diseases and in cancer. In this protein family, a conserved core domain, the so-called α-crystallin or Hsp20 domain, is flanked by highly variable, nonconserved sequences that are essential for chaperone function. Analysis of 8714 sHsps revealed a broad variation of primary sequences within the superfamily as well as phyla-dependent differences. Significant variations were found in the number of sHsps per genome, their amino acid composition, and the length distribution of the different sequence parts. Reconstruction of the evolutionary tree for the sHsp superfamily shows that the flanking regions fall into several subgroups, indicating that they were remodeled several times in parallel but independent of the evolution of the α-crystallin domain. The evolutionary history of sHsps is thus set apart from that of other protein families in that two exon boundary-independent strategies are combined: the evolution of the conserved α-crystallin domain and the independent evolution of the N- and C-terminal sequences. This scenario allows for increased variability in specific small parts of the protein and thus promotes functional and structural differentiation of sHsps, which is not reflected in the general evolutionary tree of species.

The dynamic genome of Hydra.

The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann-Mangold organizer, pluripotency genes and the neuromuscular junction.

Combined genomic and proteomic approaches identify gene clusters involved in anaerobic 2-methylnaphthalene degradation in the sulfate-reducing enrichment culture N47.

The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydrocarbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductive ring cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO(2). The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms), composed of alpha-, beta-, and gamma-subunits that are encoded by the genes nmsABC. Located upstream of nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe(4)S(4)] cluster sequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradation within the denitrifiers "Aromatoleum aromaticum" EbN1, Azoarcus sp. strain T, and Thauera aromatica. Another contiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes that were found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that were supposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetyl-CoA and CO(2). Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the gene product to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work provides the first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation.

Modification of the in vitro release profile of Cetrorelix by complexation with biophilic partners.

Cetrorelix is a GnRH antagonist of the third generation. Its manifold therapeutic potential requires the adjustment of its resorption rates and effect profiles. The method of non-covalent complexation with suitable partner molecules enables the development of customized depot formulations. Investigating new partners and synthesis methods for Cetrorelix complexes we focused on maximal biocompatibility of the complexes. Compared to traditional depot forms the application of complexes aims at decreased aggregation of the peptide and increased biophily of the depots. The pharmacological properties of the new Cetrorelix complexes were analyzed by standardized dynamical in vitro liberation experiments. A new pharmacokinetic model has been developed and successfully applied for the quantitative analysis of the liberation profiles. With aromatic carboxylic acids and dipeptides we could synthesize stable complexes that have nearly linear release characteristics in aggregating environments close to in vivo conditions. The release rates were specific and very different for the complex partners. Thus several complexes have a great potential for a linear, characteristic release of the peptide in vivo and can be the basis for new depot forms for Cetrorelix.