The m gamma delta-1 element, a small gamma delta (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing.

Transposon gamma delta (Tn1000), a 6-kb member of the Tn3 family, is widely used for plasmid mutagenesis. A 1.8-kb derivative of gamma delta was constructed that contains the kan gene from Tn5 and the resolution (res) site from gamma delta cloned between 40-bp inverted repeats of gamma delta's delta (delta) end. This element, named m gamma delta-1, lacks the genes encoding transposase and resolvase, and therefore depends on its host to supply transposition and resolution functions. Thus, in strains lacking gamma delta, m gamma delta-1 will not transpose. The m gamma delta-1 element is shown to be useful for mutagenesis of plasmids, DNA sequencing, and allele replacement (in Streptomyces avermitilis).     

A low molecular weight alkaline proteinase fromConidiobolus coronatus

Summary An extracellular, low molecular weight alkaline proteinase (alkaline proteinase B) has been purified to homogeneity from the culture filtrate ofConidiobolus coronatus (NCIM 1238). A 12-fold purification was achieved with a specific activity of 29,760 u/mg. The enzyme had an optimum pH and temperature of 9.7 and 45°C respectively. It was most active towards casein and had a molecular weight of 6,800, the lowest reported so far. It was stable between pH 6.5–7.5. Alkaline proteinase B is a serine proteinase. It showed an esterolytic activity on N-benzoyl-L-tyrosine ethyl ester (BTEE) and was successfully used to resolve the racemic mixture of D, L-phenylalanine and D,L-phenylglycine and can thus potentially replace subtilisin Carlsberg in resolving the racemic mixture of amino acids.     

beta-Glucosidase of Penicillium funiculosum. I. Purification

A strain of Penicillium funiculosum, isolated in this laboratory, produced in high yield both endo- and exo-glucanases and beta-glucosidases, which were suitable for the saccharification of cellulosic materials. The isolation of the beta-glucosidase of this organism, which differs from other beta-glucosidases of fungi in its substrate specificity, by preparative electrophoresis, is described in this article. The organism was grown on a lactose-casein medium and the culture filtrate concentrated by ammonium sulfate precipitation and dialysis. Electrophoresis was carried out on large slabs of polyacrylamide gel in an anodicrun in the presence of borate at pH 7. After elution of active fractions, a cathodic run was made at pH 6.0. Two precipitations with ammonium sulfate resulted in a homogeneous enzyme (specific activity 174 IU/mg). A second isozyme was also produced by P. funiculosum on cellulose-wheat bran medium. This isozyme was purified by electrophoresis at pH 7.0 in the absence of borate and was obtained free from other isozymes of beta-glucosidase and cellulases.     

A procedure for easy removal of acrylamide gel rods from the casting glass tubes

A new method specific for the determination of subpicomole quantities of tryptophan has been developed by elaboration of the Pictet-Spengler reaction. It permitted reproducible quantitation of tryptophan in less than 1 μl of plasma ultrafiltrate or 1 mg of brain tissue. Samples deproteinized by trichloroacetic acid were boiled for 15 min with formaldehyde and potassium ferricyanide at controlled acidity, where tryptophan was converted to a single new product identified as 9-hydroxymethyl-β-carboline. It was quantitated by either direct spectrofluorometry or a reversed-phase HPLC system developed for β-carbolines. Under our conditions, peptides containing N-terminal tryptophan such as Trp-Leu and delta sleep-inducing peptide gave N-(9-hydroxymethyl-β-carboline-3-carbonyl) peptides which retained all amino acid residues except tryptophan.     

Characterization of extracellular substrate specific glucose and xylose isomerases of Chainia

Summary Specific glucose and xylose isomerases have been identified in cell-free culture filtrates of a Chainia species. Treatment with DEAE-cellulose selectively adsorbed xylose isomerase activity while only the glucose isomerase was adsorbed on CM-cellulose. Glucose isomerase was completely inhibited by xylose at 1.3 × 10^-4 M concentration. The differential identity of the extracellular glucose and xylose isomerases, unique to Chainia, is discussed.(NCL Communication 3562)     

A versatile gel casting cum electrophoresis apparatus

A simple apparatus for vertical.,in situ, polyacrylamide or agarose gel casting as well as for the subsequent electrophoresis is described. The apparatus is completely leakproof and does not require any special device like clamps, O-rings, gaskets, grease etc. for sealing. Slab gels of various thickness (0.04 to 1.0 cm) can be made and the apparatus can be used for analytical or preparative purposes. Gel rods can also be cast and run in the device. Forward as well as reverse polarity electrophoresis of a sample can be run simultaneously in the apparatus. NCL Communication No.: 3077.     

Limited Bacterial Diversity within a Treatment Plant Receiving Antibiotic-Containing Waste from Bulk Drug Production

Biological treatment of waste water from bulk drug production, contaminated with high levels of fluoroquinolone antibiotics, can lead to massive enrichment of antibiotic resistant bacteria, resistance genes and associated mobile elements, as previously shown. Such strong selection may be boosted by the use of activated sludge (AS) technology, where microbes that are able to thrive on the chemicals within the wastewater are reintroduced at an earlier stage of the process to further enhance degradation of incoming chemicals. The microbial community structure within such a treatment plant is, however, largely unclear. In this study, Illumina-based 16S rRNA amplicon sequencing was applied to investigate the bacterial communities of different stages from an Indian treatment plant operated by Patancheru Environment Technology Limited (PETL) in Hyderabad, India. The plant receives waste water with high levels of fluoroquinolones and applies AS technology. A total of 1,019,400 sequences from samples of different stages of the treatment process were analyzed. In total 202, 303, 732, 652, 947 and 864 operational taxonomic units (OTUs) were obtained at 3% distance cutoff in the equilibrator, aeration tanks 1 and 2, settling tank, secondary sludge and old sludge samples from PETL, respectively. Proteobacteria was the most dominant phyla in all samples with Gammaproteobacteria and Betaproteobacteria being the dominant classes. Alcaligenaceae and Pseudomonadaceae, bacterial families from PETL previously reported to be highly multidrug resistant, were the dominant families in aeration tank samples. Despite regular addition of human sewage (approximately 20%) to uphold microbial activity, the bacterial diversity within aeration tanks from PETL was considerably lower than corresponding samples from seven, regular municipal waste water treatment plants. The strong selection pressure from antibiotics present may be one important factor in structuring the microbial community in PETL, which may affect not only resistance promotion but also general efficiency of the waste treatment process.     

The Autodepalmitoylating Activity of APT Maintains the Spatial Organization of Palmitoylated Membrane Proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.     

Temporal sampling,resetting, and adaptation orchestrate gradient sensing in sperm

Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca²⁺ oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2–0.6 s before a Ca²⁺ response was produced. Additional molecules delivered during a Ca²⁺ response reset the cell by causing a pronounced Ca²⁺ drop that terminated the response; this reset was followed by a new Ca²⁺ rise. After stimulation, sperm adapted their sensitivity following the Weber–Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.