Validation of a Nylon-Flocked-Swab Protocol for Efficient Recovery of Bacterial Spores from Smooth and Rough Surfaces

In order to meet planetary-protection requirements, culturable bacterial spore loads are measured representatively for the total microbial contamination of spacecraft. However, the National Aeronautics and Space Administration''s (NASA''s) cotton swab protocols for spore load determination have not changed for decades. To determine whether a more efficient alternative was available, a novel swab was evaluated for recovery of different Bacillus atrophaeus spore concentrations on stainless steel and other surfaces. Two protocols for the nylon-flocked swab (NFS) were validated and compared to the present NASA standard protocol. The results indicate that the novel swab protocols recover 3- to 4-fold more (45.4% and 49.0% recovery efficiency) B. atrophaeus spores than the NASA standard method (13.2%). Moreover, the nylon-flocked-swab protocols were superior in recovery efficiency for spores of seven different Bacillus species, including Bacillus anthracis Sterne (recovery efficiency, 20%). The recovery efficiencies for B. atrophaeus spores from different surfaces showed a variation from 5.9 to 62.0%, depending on the roughness of the surface analyzed. Direct inoculation of the swab resulted in a recovery rate of about 80%, consistent with the results of scanning electron micrographs that allowed detailed comparisons of the two swab types. The results of this investigation will significantly contribute to the cleanliness control of future life detection missions and will provide significant improvement in detection of B. anthracis contamination for law enforcement and security efforts.The recent discovery of liquid water on Mars has sparked debate about the possibility of extraterrestrial life (37). Consequently, highly sensitive biosensors will be deployed onboard spacecraft like the Mars Science Laboratory (MSL), using technologies such as gas chromatographical analysis to search for the smallest traces of life (http://mars.jpl.nasa.gov/msl/mission/). Contamination of equipment by terrestrial microorganisms resulting from a lack of spacecraft cleanliness could significantly compromise the integrity of life detection missions and result in falsely positive extraterrestrial life signals. The prevention of this so-called “forward contamination” is one major goal of American and European space agencies'' planetary-protection efforts. Regular determination of a spacecraft''s bioload and the mission components throughout assembly are mandatory for detecting unacceptably high contamination that exceeds levels set by the United Nations treaty (Outer Space Treaty [11]).Modern spacecraft hardware is very susceptible to standard heat sterilization protocols, so baking the entire spacecraft, such as the Viking Lander Capsule at 111.7°C ± 1.7°C for 23 to 30 h is no longer feasible (30). Alternative cleaning and sterilization methodologies for spacecraft components prior to assembly (i.e., nonthermal plasma technologies) have been discussed (36). However, after integration, sterile hardware is exposed to a significant risk of contamination during assembly, testing, and launching operations. Because of limited access to integrated spacecraft components, the microbial cleanliness of a spacecraft and its surroundings is meticulously maintained through frequent cleaning and sterilization routines. Therefore, the regular and frequent detection of possible contaminants in the assembly environment is more important than ever.To estimate the severity of microbial contamination, the National Aeronautics and Space Administration''s (NASA''s) standard procedure focuses on aerobic, mesophilic spores (26). Briefly, surface samples are taken from spacecraft using moist cotton swabs or wipes. After an extraction procedure, the samples are subjected to a short heat shock (15 min; 80°C) to kill vegetative cells and then pour plated in Trypticase soy agar (TSA) for the enumeration of CFU. This protocol was originally developed for the Viking mission more than 3 decades ago (30) and has remained, for the most part, unchanged.Recent studies have shown that cotton swabs have acceptable recovery efficiencies for Bacillus spores (41.7%) (32) but, due to their organic nature, may raise residue problems on surfaces. Furthermore, their comparatively high DNA content could lead to false positives or inhibition should NASA one day incorporate molecular technologies into their microbial-detection protocols (7).Based on these observations, researchers are beginning to move away from cotton in favor of alternative swabs made from rayon or macrofoam (6, 18). A recent study reported high recovery efficiencies for various vegetative cells from stainless steel surfaces by applying a novel swab with a bulb-shaped head flocked with nylon fibers (12). Patented in 2004, this design facilitates the release of particulates and microbes, resulting in a significantly higher detection rate. The broad applicability of these nylon-flocked swabs (NFS) has been demonstrated by their use in various clinical studies isolating pathogens from medical environments (1, 10, 20).General studies on surface-sampling tools have clearly shown that the swab material and the extraction method are the dominant factors in spore recovery efficiencies (32). Additionally, the properties of the surface to be sampled affect sample recovery (8). For planetary-protection applications, the broad variety of novel materials used in spacecraft construction must be considered. The Mars Exploration Rover mission craft, for example, was composed of at least five kinds of surface materials (http://marsrovers.jpl.nasa.gov/overview). While the cruise stage was constructed primarily of aluminum and the aeroshell consisted of aluminum honeycomb structures, the lander itself was made of titanium and graphite composite (carbon fiber-reinforced plastic [CFRP]). The airbag and the parachutes were made of Vectran and polyester/nylon fabrics. These different materials are quite challenging for sampling tools. Accurate sampling of materials with various surface textures will require planetary-protection programs to introduce novel swab materials.To our knowledge, no investigations have been performed to compare the recovery of spores from different spacecraft surfaces. Previous studies have compared cotton and synthetic sampling materials, but only on stainless steel surfaces (19), and no studies have compared sampling methods on actual spacecraft materials (7).Recently published protocols for spore detection have been based on one specific Bacillus species and/or on one type of surface. Unfortunately, these protocols provide no insight into the effects of varying these factors (4-6, 8, 9, 14, 18), as requested by USP (United States Pharmacopeia) 1223 for validation of alternative microbial methods (3). Some of the aforementioned studies were conducted in response to B. anthracis terrorism incidents in 2001 and used B. atrophaeus as a surrogate. Consequently, information about the actual sampling efficiency of B. anthracis spores is quite limited and may vary significantly from the B. atrophaeus data.In this comprehensive study, we evaluated the novel nylon-flocked swab and a corresponding protocol to recover Bacillus spores from five different spacecraft-related surfaces. It should be noted that although stainless steel served as the standard test surface, it is not a predominant material in spacecraft; however, since the majority of previous (sampling) studies were performed on stainless steel, it represents a universally recognized carrier and also serves as a conservative proxy for the average roughness of the materials used in space science.Our nylon-flocked-swab protocol was validated with respect to accuracy, precision, limit of detection, linearity, and robustness (3). Moreover, its specificity was determined by applying spores of seven different Bacillus species, including the avirulent, attenuated strain Bacillus anthracis Sterne, and by comparing the resulting recovery efficiencies. The results in this communication will significantly contribute to planetary-protection protocols and could also be of high interest for public health issues.     

Electrical protein array chips for the detection of staphylococcal virulence factors

A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.     

CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice

Despite extensive investigations of Cbl‐interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85^Δex2) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85^Δex2 animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post‐synaptic compartment of striatal neurons in which it co‐clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85^Δex2 mice.     

Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic β-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9–39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Åresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous α-helix from Thr¹³ to Val³³ when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.     

Analysis of Lipid Export in Hydrocarbonoclastic Bacteria of the Genus Alcanivorax: Identification of Lipid Export-Negative Mutants of Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9

Triacylglycerols (TAGs), wax esters (WEs), and polyhydroxyalkanoates (PHAs) are the major hydrophobic compounds synthesized in bacteria and deposited as cytoplasmic inclusion bodies when cells are cultivated under imbalanced growth conditions. The intracellular occurrence of these compounds causes high costs for downstream processing. Alcanivorax species are able to produce extracellular lipids when the cells are cultivated on hexadecane or pyruvate as the sole carbon source. In this study, we developed a screening procedure to isolate lipid export-negative transposon-induced mutants of bacteria of the genus Alcanivorax for identification of genes required for lipid export by employing the dyes Nile red and Solvent Blue 38. Three transposon-induced mutants of A. jadensis and seven of A. borkumensis impaired in lipid secretion were isolated. All isolated mutants were still capable of synthesizing and accumulating these lipids intracellularly and exhibited no growth defect. In the A. jadensis mutants, the transposon insertions were mapped in genes annotated as encoding a putative DNA repair system specific for alkylated DNA (Aj17), a magnesium transporter (Aj7), and a transposase (Aj5). In the A. borkumensis mutants, the insertions were mapped in genes encoding different proteins involved in various transport processes, like genes encoding (i) a heavy metal resistance (CZCA2) in mutant ABO_6/39, (ii) a multidrug efflux (MATE efflux) protein in mutant ABO_25/21, (iii) an alginate lyase (AlgL) in mutants ABO_10/30 and ABO_19/48, (iv) a sodium-dicarboxylate symporter family protein (GltP) in mutant ABO_27/29, (v) an alginate transporter (AlgE) in mutant ABO_26/1, or (vi) a two-component system protein in mutant ABO_27/56. Site-directed MATE, algE, and algL gene disruption mutants, which were constructed in addition, were also unable to export neutral lipids and confirmed the phenotype of the transposon-induced mutants. The putative localization of the different gene products and their possible roles in lipid excretion are discussed. Beside this, the composition of the intra- and extracellular lipids in the wild types and mutants were analyzed in detail.Almost all prokaryotes synthesize lipophilic storage substances as an integral part of their metabolism under limited nitrogen or phosphorus conditions if there is an excess of a suitable carbon source at the same time. The accumulated storage lipids serve as energy and carbon sources during starvation periods, and they are mobilized again under conditions of carbon and energy deficiency. The majority of the members of many genera synthesize hydrophobic polymers, such as poly(3-hydroxybutyrate) (PHB) or other types of polyhydroxyalkanoates (PHAs), whereas the accumulation of triacylglycerols (TAGs; trioxoesters of glycerol and long-chain fatty acids [FAs]) or wax esters (WEs; oxoesters of primary long-chain fatty acids and primary long-chain fatty alcohols) occurs in fewer prokaryotes (66). TAG accumulation has been reported for species of the genera Streptomyces, Mycobacterium, Nocardia, Rhodococcus (4, 6, 65), and recently also Alcanivorax and other hydrocarbonoclastic marine bacteria (32). Accumulation of WEs has been frequently reported for species of the genus Acinetobacter (66) but also for marine bacteria, such as Marinobacter (50) and Alcanivorax (11, 32).In general, the accumulation of at least one type of these compounds occurs intracellularly under imbalanced growth conditions in almost all prokaryotes. The localization of neutral lipids in marine organisms is not restricted to the cell cytoplasm, as extracellular lipid deposition has been shown in studies with Alcaligenes sp. PHY9 and Pseudomonas nautica (24). The production of extracellular wax esters by Alcanivorax jadensis T9 growing on hexadecane was described a few years ago (11). Species of the genus Alcanivorax belong to an unusual group of marine hydrocarbon-degrading bacteria, which have been recognized and described over the past few years and were shown to play an important role in the biological removal of petroleum hydrocarbons from contaminated sites (69). Species of the genus Alcanivorax are, like some species of the genera Neptunomonas (27) and Marinobacter (23), marine hydrocarbon-degrading bacteria. Moreover, Alcanivorax and related bacteria constitute the group of obligate hydrocarbonoclastic marine bacteria (OHCB), which exhibit a narrow range of utilizable carbon sources (obligate hydrocarbon utilization), with only a few species being able to metabolize substrates other than hydrocarbons (69). Alcanivorax borkumensis SK2 became a model strain of OHCB, and its importance and pivotal role in hydrocarbon biodegradation have recently been emphasized (33). The predominance of A. borkumensis in early stages of petroleum degradation has also been reported in microcosm studies as well as for a field-scale experiment (26).From a biotechnological point of view, the production of extracellular lipids is important. Secretion of lipophilic products into the culture medium rather than its intracellular accumulation can significantly reduce the costs of product recovery. Another advantage is that the production of WEs and TAGs would not be directly limited by cell density or cell volume. Until now, the mechanism responsible for the export of lipids in bacteria of the genus Alcanivorax or other bacteria had not been known. In this study, we report on a screening procedure to select mutants defective in lipid export for identification of the gene(s) involved in the export mechanism. After transposon-induced mutagenesis we found different mutants which were not able to export TAGs (mutants of A. borkumensis) when the cells were cultivated in the presence of pyruvate as the sole carbon source. Mutants of A. jadensis defective in export of WEs and/or wax diesters (DE) were also identified. The possible influences of the gene products on the export mechanism in Alcanivorax species were analyzed and are discussed.     

Genome-based phylogenetic analysis of Streptomyces and its relatives

MotivationStreptomyces is one of the best-studied genera of the order Actinomycetales due to its great importance in medical science, ecology and the biotechnology industry. A comprehensive, detailed and robust phylogeny of Streptomyces and its relatives is needed for understanding how this group emerged and maintained such a vast diversity throughout evolution and how soil-living mycelial forms (e.g., Streptomyces s. str.) are related to parasitic, unicellular pathogens (e.g., Mycobacterium tuberculosis) or marine species (e.g., Salinispora tropica). The most important application area of such a phylogenetic analysis will be in the comparative re-annotation of genome sequences and the reconstruction of Streptomyces metabolic networks for biotechnology.MethodsClassical 16S-rRNA-based phylogenetic reconstruction does not guarantee to produce well-resolved robust trees that reflect the overall relationship between bacterial species with widespread horizontal gene transfer. In our study we therefore combine three whole genome-based phylogenies with eight different, highly informative single-gene phylogenies to determine a new robust consensus tree of 45 Actinomycetales species with completely sequenced genomes.ResultsNone of the individual methods achieved a resolved phylogeny of Streptomyces and its relatives. Single-gene approaches failed to yield a detailed phylogeny; even though the single trees are in good agreement among each other, they show very low resolution of inner branches. The three whole genome-based methods improve resolution considerably. Only by combining the phylogenies from single gene-based and genome-based approaches we finally obtained a consensus tree with well-resolved branches for the entire set of Actinomycetales species. This phylogenetic information is stable and informative enough for application to the system-wide comparative modeling of bacterial physiology.     

Metabonomic fingerprints of fasting plasma and spot urine reveal human pre-diabetic metabolic traits

Impaired glucose tolerance (IGT) which precedes overt type 2 diabetes (T2DM) for decades is associated with multiple metabolic alterations in insulin sensitive tissues. In an UPLC-qTOF-mass spectrometry-driven non-targeted metabonomics approach we investigated plasma as well as spot urine of 51 non-diabetic, overnight fasted individuals aiming to separate subjects with IGT from controls thereby identify pathways affected by the pre-diabetic metabolic state. We could clearly demonstrate that normal glucose tolerant (NGT) and IGT subjects clustered in two distinct groups independent of the investigated metabonome. These findings reflect considerable differences in individual metabolite fingerprints, both in plasma and urine. Pre-diabetes associated alterations in fatty acid-, tryptophan-, uric acid-, bile acid-, and lysophosphatidylcholine-metabolism, as well as the TCA cycle were identified. Of note, individuals with IGT also showed decreased levels of gut flora-associated metabolites namely hippuric acid, methylxanthine, methyluric acid, and 3-hydroxyhippuric acid. The findings of our non-targeted UPLC-qTOF-MS metabonomics analysis in plasma and spot urine of individuals with IGT vs NGT offers novel insights into the metabolic alterations occurring in the long, asymptomatic period preceding the manifestation of T2DM thereby giving prospects for new intervention targets.     

Contribution of charged and polar residues for the formation of the E1–E2 heterodimer from Hepatitis C Virus

The transmembrane domains of the envelope glycoprotein E1 and E2 have crucial multifunctional roles in the biogenesis of hepatitis C virus. We have performed molecular dynamics simulations to investigate a structural model of the transmembrane segments of the E1–E2 heterodimer. The simulations support the key role of the Lys370–Asp728 ion pair for mediating the E1–E2 heterodimerization. In comparison to these two residues, the simulation results also reveal the differential effect of the conserved Arg730 residue that has been observed in experimental studies. Furthermore, we discovered the formation of inter-helical hydrogen bonds via Asn367 that stabilize dimer formation. Simulations of single and double mutants further demonstrate the importance of the ion-pair and polar interactions between the interacting helix monomers. The conformation of the E1 fragment in the simulation of the E1–E2 heterodimer is in close agreement with an NMR structure of the E1 transmembrane segment. The proposed model of the E1–E2 heterodimer supports the postulated cooperative insertion of both helices by the translocon complex into the bilayer.