β-Lactamases from Actinopolyspora halophila,an extremely halophilic actinomycete

Actinopolyspora halophila, an extremely halophilic actinomycete, produced both cellular and exocellular -lactamases when grown in liquid media containing 20% (w/v) NaCl. Maximal exocellular -lactamase production occurred at 48 h growth and exceeded cellular enzyme levels 7-fold. Maximal cellular -lactamase was observed at 72 h as cultures achieved full growth.Both -lactamases were purified to molecular homogeneity by a sequence involving gel filtration on Bio-Gel P-100, DEAE Bio-Gel A chromatography, preparative isoelectric focusing, and gel filtration on Sephadex G-75. Cellular -lactamase was purified 99-fold with 12% recovery and had a molecular weight of 42,200, and an isoelectric point of 4.15. Exocellular -lactamase was 12-fold purified with 1.2% recovery of initial activity and had a molecular weight of 38,000 and an isoelectric point of 3.85. Its specific activity was 7-fold greater than that of the cellular enzyme.A variety of penicillin and cephalosporin substrates were degraded by both enzymes. While the cellular -lactamase degraded phenoxymethylpenicillin, methicillin, and cephaloglycin most efficiently, the exocellular enzyme was most active against methicillin, carbenicillin, ampicillin, and cephalosporin C. Both enzymes were stimulated and protected from thermal deactivation by NaCl but not KCl or MgCl₂-Neither enzyme was inhibited by iodine.Abbreviations IEF Isoelectric focusing - SDS-PAGE sodium dodecylsulphate-polyacrylamide electrophoresis - PEG polyethyleneglycol - DTT dithiothreitol - DEAE diethylaminoethyl Issued as NRCC 25164     

Purification and analysis of an extremely halophilic β-galactosidase from Haloferax alicantei

As a first step in the development of a reporter system for gene expression in halophilic archaea, a β-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180±20 kDa in size, consisting of two monomers (each 78±3 kDa). It cleaves several different β-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has β-d-fucosidase activity. No β-glucosidase, β-arabinosidase or β-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.     

Response of human keratinocytes to extremely low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine

Since alkylating agents are widely present in the environment and constitute a continuous challenge to genome integrity, cells and organisms have developed defense mechanisms to remove such lesions. We monitored the response of human keratinocytes to a very low concentration of a methylating agent, namely 2.5 nM N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The effect of a 60-min exposure of quiescent cells to 2.5 nM MNNG was studied in terms of DNA integrity, poly(ADP-ribose) metabolism, clonogenic survival and DNA synthesis. We observed two waves of DNA strand break formation and resealing. Interestingly, the amount of DNA strand breaks in exposed cells was lower than in unexposed control cells. This phenomenon was also observed when cells were exposed to MNNG in the presence of a protein synthesis inhibitor, or when they were maintained on ice during the treatment. A dose of 2.5 nM MNNG stimulated poly(ADP-ribose) turnover, reduced the intracellular NAD⁺ content, stimulated DNA synthesis and caused a remarkable increase in clonogenic survival. Thus, the cellular responses to extremely low concentrations of MNNG differ sharply from those observed at higher doses of this carcinogen. We conclude that the very low dose response cannot be extrapolated from usual dose-response analyses.     

Biofilm development in the extremely acidophilic archaeon ‘Ferroplasma acidarmanus’ Fer1

‘Ferroplasma acidarmanus’ Fer1 is an iron-oxidizing extreme acidophile isolated from the Iron Mountain mine, California, USA. This archaeon is predominantly found in biofilm-associated structures in the environment, and produces two distinct biofilm morphologies. Bioinformatic analysis of the ‘F. acidarmanus’ Fer1 genome identified genes annotated as involved in attachment and biofilm formation. No putative quorum sensing signaling genes were identified and no N-acyl homoserine lactone-like compounds were found in ‘F. acidarmanus’ Fer1 biofilm supernatant. Scanning confocal microscopy analysis of biofilm development on the surface of pyrite demonstrated the temporal and spatial development of biofilm growth. Furthermore, two-dimensional polyacrylamide gel electrophoresis was used to examine differential protein expression patterns between biofilm and planktonic populations. Ten up-regulated proteins were identified that included six enzymes associated with anaerobic growth, suggesting that the dominating phenotype in the mature biofilm was associated with anaerobic modes of growth. This report increases our knowledge of the genetic and proteomic basis of biofilm formation in an extreme acidophilic archaeon.     

Innate immune deficiency of extremely premature neonates can be reversed by interferon-γ

Background

Bacterial sepsis is a major threat in neonates born prematurely, and is associated with elevated morbidity and mortality. Little is known on the innate immune response to bacteria among extremely premature infants.

Methodology/Principal Findings

We compared innate immune functions to bacteria commonly causing sepsis in 21 infants of less than 28 wks of gestational age, 24 infants born between 28 and 32 wks of gestational age, 25 term newborns and 20 healthy adults. Levels of surface expression of innate immune receptors (CD14, TLR2, TLR4, and MD-2) for Gram-positive and Gram-negative bacteria were measured in cord blood leukocytes at the time of birth. The cytokine response to bacteria of those leukocytes as well as plasma-dependent opsonophagocytosis of bacteria by target leukocytes was also measured in the presence or absence of interferon-γ. Leukocytes from extremely premature infants expressed very low levels of receptors important for bacterial recognition. Leukocyte inflammatory responses to bacteria and opsonophagocytic activity of plasma from premature infants were also severely impaired compared to term newborns or adults. These innate immune defects could be corrected when blood from premature infants was incubated ex vivo 12 hrs with interferon-γ.

Conclusion/Significance

Premature infants display markedly impaired innate immune functions, which likely account for their propensity to develop bacterial sepsis during the neonatal period. The fetal innate immune response progressively matures in the last three months in utero. Ex vivo treatment of leukocytes from premature neonates with interferon-γ reversed their innate immune responses deficiency to bacteria. These data represent a promising proof-of-concept to treat premature newborns at the time of delivery with pharmacological agents aimed at maturing innate immune responses in order to prevent neonatal sepsis.     

Modeling protein–nucleic acid complexes with extremely large conformational changes using Flex-LZerD

Proteins and nucleic acids are key components in many processes in living cells, and interactions between proteins and nucleic acids are often crucial pathway components. In many cases, large flexibility of proteins as they interact with nucleic acids is key to their function. To understand the mechanisms of these processes, it is necessary to consider the 3D atomic structures of such protein–nucleic acid complexes. When such structures are not yet experimentally determined, protein docking can be used to computationally generate useful structure models. However, such docking has long had the limitation that the consideration of flexibility is usually limited to small movements or to small structures. We previously developed a method of flexible protein docking which could model ordered proteins which undergo large-scale conformational changes, which we also showed was compatible with nucleic acids. Here, we elaborate on the ability of that pipeline, Flex-LZerD, to model specifically interactions between proteins and nucleic acids, and demonstrate that Flex-LZerD can model more interactions and types of conformational change than previously shown.     

Enrichment of extremely noisy high-throughput screening data using a naïve Bayes classifier

The noise level of a high-throughput screening (HTS) experiment depends on various factors such as the quality and robustness of the assay itself and the quality of the robotic platform. Screening of compound mixtures is noisier than screening single compounds per well. A classification model based on na?ve Bayes (NB) may be used to enrich such data. The authors studied the ability of the NB classifier to prioritize noisy primary HTS data of compound mixtures (5 compounds/well) in 4 campaigns in which the percentage of noise presumed to be inactive compounds ranged between 81% and 91%. The top 10% of the compounds suggested by the classifier captured between 26% and 45% of the active compounds. These results are reasonable and useful, considering the poor quality of the training set and the short computing time that is needed to build and deploy the classifier.     

F.G. syndrome: a rare and/or extremely polymorphic syndrome?

We report a case of mental retardation associated with multiple congenital anomalies suggesting an F.G. syndrome. We discuss problems concerning genetic counselling and the management of future pregnancies. Unfortunately, no concrete strategy, concerning prenatal diagnosis, can be proposed.     

Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

The characterization of C-phycocyanin from an extremely halo-tolerant blue–green alga, Coccochloris elabens

C-Phycocyanin was isolated and purified from a uni-algal culture of an extremely halo-tolerant blue-green alga, Coccochloris elabens. This alga can be grown under laboratory conditions in 25% (w/v) NaCl. Purified halophile phycocyanin was characterized by amino acid analysis and the measurement of sedimentation velocity, fluorescence polarization and immunodiffusion as a function of protein concentration, pH and ionic strength. The results were compared with those of studies of phycocyanin isolated from Plectonema calothricoides and from several other sources. The states of aggregation previously characterized as being present in other C-phycocyanins, monomer, trimer and hexamer, were present in halophile phycocyanin and were characterized as antigenically related to all C-phycocyanins tested. The equilibrium between 3S monomer and 11S hexamer at low concentrations in halophile phycocyanin was quantitatively similar to that for other phycocyanins. The effect of pH and ionic strength on the 6S (trimer) and 11S (hexamer) aggregation of halophile phycocyanin was markedly salt-dependent and the relative amount of each aggregate in the presence of 2m-NaCl was like that of C-phycocyanin from mesophiles, in the absence of additional salt. In antigenic relationship and aggregation properties, the phycocyanin from C. elabens appeared to be most closely related to that isolated from the thermophilic blue-green alga, Synechococcus lividus. Amino acid content of the halophile phycocyanin indicated the presence of a significantly larger number of acidic residues than that found in mesophiles. Explanations of the properties of the halophile protein require consideration of a strong contribution of hydrophobic forces and utilize both charge-shielding and salting-out effects.     

Use of extremely short F?rster resonance energy transfer probes in real-time polymerase chain reaction

Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed ‘probe-luring’ sequence at their 5′ ends. Hybridization of this sequence to a complementary ‘anchoring’ tail introduced at the 3′ end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a ‘universal’ probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.     

Phytoplankton of the extremely acidic mining lakes of Lusatia (Germany) with pH ≤3

Most of the flooded, open-cast lignite mining lakes of Lusatia (Germany) impacted by the oxidation of iron sulphides (pyrite and marcasite) are extremely acidic. Of 32 lakes regularly studied from 1995 to 1998, 14 have a pH <3 (median pH 2.3–2.9). These lakes are typically buffered by high concentrations of Fe (III) and have high conductivity (1000–5000 S cm^–1). Concentrations of dissolved inorganic carbon (DIC) and phosphorus are typically extremely low. These factors result in a very different environment for algae than found in neutral and acid-rain impacted lakes. The planktonic algal flora is generally dominated by flagellates belonging to genera of Chlorophyta (Chlamydomonas), Heterokontophyta of the class Chrysophyceae (Ochromonas, Chromulina), Cryptophyta (Cyathomonas) and Euglenophyta (Lepocinclis, Euglena mutabilis). Near-spherical non-motile Chlorophyta (Nanochlorum sp.), Heterokontophyta of the class Bacillariophyceae (Eunotia exigua, Nitzschia), Dinophyta (Gymnodinium, Peridinium umbonatum), other Chlorophyta (Scourfieldia cordiformis) and Cryptophyta (Rhodomonas minuta) are also found.     

Application of β-glucuronidase (GusA) as an effective reporter for extremely acidophilic Acidithiobacillus ferrooxidans

Applied Microbiology and Biotechnology - Acidithiobacillus ferrooxidans is a model organism for investigating metal sulfide bioleaching. The regulatory mechanism of gene expression by metabolizing...     

Fervidobacterium islandicum sp. nov., a new extremely thermophilic eubacterium belonging to the “Thermotogales”

An extremely thermophilic anaerobic fermentative eubacterium growing at temperatures between 50 and 80°C (opt.: 65°C) was isolated from an Icelandic hot spring. The cells were Gram-negative motile rods, about 1.8 m in length, and 0.6 m in width occurring singly and in pairs. About 50% of the cells formed large spheroids at one end similar to Fervidobacterium nodosum. The new isolate H 21 differed from Fervidobacterium nodosum by a 6 mol % higher GC-content of its DNA (41 mol %), its ability to grow on cellulose, and insignificant DNA homology. The lipids of isolate H 21 were similar to that of members of Thermotogales. 16S rRNA sequencing of isolate H 21 and Fervidobacterium nodosum indicated (a) that isolate H 21 represents a new species of the genus Fervidobacterium which we name Fervidobacterium islandicum and (b) that the genus Fervidobacterium belongs to the Thermotogales branch.     

An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous β-galactosidase gene

Three phenotypically stable mutants of the extremely thermophilic archaeon Sulfolobus solfataricus have been isolated by screening for β-galactosidase negative colonies on plates with X-Gal (5-bromo-4-chloro-3-indolyl-(3-d-galactopyranoside). From one of these mutants an insertion element, designated ISC1217, was isolated and characterized. Sequence analysis of ISC1217 and of the regions adjacent to the insertion site in the β-galactosidase gene revealed features typical of a transposable element: ISC1217 contained terminal inverted repeats and was flanked by a direct repeat of 6 bp. The 1147 by sequence contained an open reading frame encoding a putative protein of 354 amino acid residues and, overlapping this, two smaller open reading frames on the opposite strand. There were approximately 8 copies of the insertion element in the S. solfataricus genome. ISC1217 did not cross-hybridize with DNA of other Sulfolobus species. All three independently isolated β-galactosidase mutants of S. solfataricus arose by transposition of ISC1217 or a related element.     

Production of halostable β-mannanase and β-mannosidase by strain NN, a new extremely halotolerant bacterium

An extremely halotolerant mannan-degrading bacterium (strain NN) was isolated from the Great Salt Lake, Utah, USA. Strain NN grew at salinities from 0 to 20% NaCl with optimal growth at 0% NaCl. When grown on 0.2% (w/v) locust bean gum as the carbon source at 10% NaCl, both β-mannanase and β-mannosidase activities were produced. β-Mannosidase activity was shown to be cell-associated, while at least 23% of the total β-mannanase activity was extracellular. The optimum temperature and pH for β-mannanase activity were 70 °C and 7.6, and for β-mannosidase 25 °C and 7.0. The β-mannanase system retained full activity after 24 h of incubation at 60 °C and 10% NaCl. β-Mannanase activity was maximal at 1% NaCl and β-mannosidase activity at 0.5% NaCl. Despite these low salinity optima, 50% and 100% respectively of the initial β-mannanase and β-mannosidase activities remained after 48 h of incubation at 20% NaCl, indicating a high degree of halostability. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed the presence of at least eight different mannan-degrading proteins in the cell-free culture supernatant of cultures grown on locust bean gum. Received: 19 March 1998 /  Received revision: 8 June 1998 / Accepted: 14 June 1998