Analysis of the flagellar bending waves of ejaculated ram sperm

The variability of flagellar movement, illustrated by the highly heterogeneous nature of the ejaculated sperm population of the ram, was analyzed by the use of a stroboscopic technique and an adapted microphotographic 24 X 36 camera system. The multiple-moving-exposures (MME) records give very distinct successive sequences of the flagellar beats and are particularly suitable for the analysis of bend development and propagation along the tail. With this technique, the parameters of the flagellar bending waves of ejaculated ram sperm have been determined. Most of the sperm have planar flagellar beatings; few are rolling under the conditions of observation. The trajectories of the gametes are mostly linear; nevertheless, some have circular paths. The analysis of bending has been focused on two examples for which the difference in the progressiveness ratio was maximum. The circular pathways for ram spermatozoa are linked to an asymmetry between principal and reverse bend probably induced by differences in wave propagation evidenced along the flagellum. A typical sperm flagellar movement may be related either to the conditions of the observations or to some differences in the maturation process of the sperm.     

Metabolism of arylsulphonates by micro-organisms

1. Species of Pseudomonas capable of degrading arylsulphonates and detergents of the alkylbenzenesulphonate type were isolated from sewage and river water. 2. Benzenesulphinate, benzenesulphonate and toluene-p-sulphonate were rapidly degraded by these organisms with the release of the sulphonate group as sulphite; detergent homologues with a chain length up to 16 carbon atoms (4-n-hexadecyl-benzenesulphonate) also released sulphite. Sulphite oxidation to sulphate in the medium can occur non-enzymically. 3. Growth on benzenesulphonate and toluene-p-sulphonate elicited a catechol 2,3-oxygenase, which effected a ;meta' cleavage of the ring. The metabolic route for benzenesulphonate was determined as: benzenesulphonate-->catechol-->2-hydroxymuconic semialdehyde-->formate and 4-hydroxy-2-oxovalerate-->acetaldehyde and pyruvate; the enzymes catalysing these steps were all inducible. 4. Toluene-p-sulphonate was degraded via 2-hydroxy-5-methylmuconic semialdehyde to formate and 4-hydroxy-2-oxohexanoate and the latter was cleaved to propionaldehyde and pyruvate. Propionaldehyde and propionate were oxidized rapidly by toluene-p-sulphonate-grown cells but slowly by fumarate-grown organisms. 5. The specificity of the catechol 2,3-oxygenase induced by the arylsulphonates, towards catechol and the methylcatechols, varied during the purification and suggested that 3-methylcatechol was probably oxidized by a separate enzyme. Detergents of the alkylbenzenesulphonate type also induced a catechol 2,3-oxygenase in these bacteria. 6. A few isolates, after growth on benzenesulphonate, opened the ring of catechol by an ;ortho' route to form cis-cis-muconate. The enzymes to degrade this intermediate to beta-oxoadipate were also present in induced cells.     

Utilization of organophosphonates by environmental micro-organisms

A survey of the utilization by environmental micro-organisms of a range of compounds containing the carbon–phosphorus (C–P) bond was carried out. Elective culture studies indicated that 15 of 19 alkylphosphonates tested served only as a sole source of phosphorus for microbial growth. Their metabolism did not lead to the extracellular release of inorganic phosphate. However, four organophosphonates—phosphonoacetate, phosphonoalanine, 2-aminoethylphosphonate and phosphonomycin—supported microbial growth when supplied as either a phosphorus source or as a carbon and energy source, with near-quantitative inorganic phosphate release. Four of five aminoalkylphosphonates tested were also utilized as a nitrogen source in the presence of 1 mmol l⁻¹ inorganic phosphate. In a subsequent screening programme, 99% of bacterial isolates tested were able to utilize 2-aminoethylphosphonate as a sole phosphorus source, 61% as a nitrogen source, 10% as a source of nitrogen and phosphorus, and 2% as a source of carbon, nitrogen and phosphorus ; 2% of isolates used phosphonoalanine as a nitrogen source. These results suggest that the uptake and metabolism of organophosphonates by bacteria is less `tightly' regulated by phosphorus starvation than has previously been supposed.     

Properties of flagellar rigor waves formed by abrupt removal of adenosine triphosphate from actively swimming sea urchin sperm

Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 µM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 µM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.     

Computer simulation of flagellar movement VIII: coordination of dynein by local curvature control can generate helical bending waves

Computer simulations have been carried out with a model flagellum that can bend in three dimensions. A pattern of dynein activation in which regions of dynein activity propagate along each doublet, with a phase shift of approximately 1/9 wavelength between adjacent doublets, will produce a helical bending wave. This pattern can be termed "doublet metachronism." The simulations show that doublet metachronism can arise spontaneously in a model axoneme in which activation of dyneins is controlled locally by the curvature of each outer doublet microtubule. In this model, dyneins operate both as sensors of curvature and as motors. Doublet metachronism and the chirality of the resulting helical bending pattern are regulated by the angular difference between the direction of the moment and sliding produced by dyneins on a doublet and the direction of the controlling curvature for that doublet. A flagellum that is generating a helical bending wave experiences twisting moments when it moves against external viscous resistance. At high viscosities, helical bending will be significantly modified by twist unless the twist resistance is greater than previously estimated. Spontaneous doublet metachronism must be modified or overridden in order for a flagellum to generate the planar bending waves that are required for efficient propulsion of spermatozoa. Planar bending can be achieved with the three-dimensional flagellar model by appropriate specification of the direction of the controlling curvature for each doublet. However, experimental observations indicate that this "hard-wired" solution is not appropriate for real flagella.     

Dimethyl sulphoxide reduction by micro-organisms.

Dimethyl sulphoxide (DMSO) was reduced to dimethyl sulphide by a wide variety of micro-organism, including prokaryotes and eukaryotes, aerobes and anaerobes. Dimethyl sulphone was not reduced by any of the organisms tested. Cell-free extracts of Escherichia coli reduced DMSO using reduced pyridine nucleotides as electron donors. Activity was greater in anaerobically grown cells than in those grown aerobically. Two other sulphoxides, methionine sulphoxide and tetramethylene sulphoxide, substantially inhibited DMSO reduction by extracts. Mutants of E. coli, which were unable to reduce biotin sulphoxide to biotin, were tested for their ability to reduce DMSO in whole cells and extracts. These mutants were in four different gene loci, bisA to bisD. DMSO reductase activity of the mutants was generally less than that of the wild-type strain, and activity depended upon the gene locus involved, the growth medium and the growth conditions. Only the bisA mutant had very low activity under all conditions. All of the bis mutants were able to grow using methionine sulphoxide as a sulphur source, indicating that biotin sulphoxide and methionine sulphoxide are reduced by different enzyme systems. DMSO may be reduced by both of these enzyme systems.     

Protein phosphorylation is a key event of flagellar disassembly revealed by analysis of flagellar phosphoproteins during flagellar shortening in Chlamydomonas

Cilia are disassembled prior to cell division, which is proposed to regulate proper cell cycle progression. The signaling pathways that regulate cilia disassembly are not well-understood. Recent biochemical and genetic data demonstrate that protein phosphorylation plays important roles in cilia disassembly. Here, we analyzed the phosphoproteins in the membrane/matrix fraction of flagella undergoing shortening as well as flagella from steady state cells of Chlamydomonas. The phosphopeptides were enriched by a combination of IMAC and titanium dioxide chromatography with a strategy of sequential elution from IMAC (SIMAC) and analyzed by tandem mass spectrometry. A total of 224 phosphoproteins derived from 1296 spectral counts of phosphopeptides were identified. Among the identified phosphoproteins are flagellar motility proteins such as outer dynein arm, intraflagellar transport proteins as well as signaling molecules including protein kinases, phosphatases, G proteins, and ion channels. Eighty-nine of these phosphoproteins were only detected in shortening flagella, whereas 29 were solely in flagella of steady growing cells, indicating dramatic changes of protein phosphorylation during flagellar shortening. Our data indicates that protein phosphorylation is a key event in flagellar disassembly, and paves the way for further study of flagellar assembly and disassembly controlled by protein phosphorylation.     

Degradation of 3-chlorobenzoate by thermophilic micro-organisms

Thermophilic (75°C), anaerobic biodegradation of chlorobenzoates was investigated using different inocula from geothermal and non-geothermal environments. Microbial dehalogenation of 3-chlorobenzoate (0·5 mmol l⁻¹) was achieved by two mixed cultures growing anaerobically at 75°C. One culture consisted of a facultative anaerobe and two obligate anaerobes, one of which was a methanogen, isolated from terrestrial sediments from hot springs in New Zealand. The other culture, derived from a non-geothermal environment, consisted of a Clostridium spp. and a non-spore-forming obligate anaerobe. No degradation of either 2-chlorobenzoate or 4-chlorobenzoate was achieved by these thermophilic cultures over the same time period. This is the first reported biotransformation of this chlorinated aromatic at a temperature of 75°C.     

Degradation of naphthenic acids by sediment micro-organisms

AIMS: Naphthenic acids (NAs) are naturally occurring, linear and cyclic carboxylic surfactants associated with the acidic fraction of petroleum. NAs account for most of the acute aquatic toxicity of oil sands process-affected water (OSPW). The toxicity of OSPW can be reduced by microbial degradation. The aim of this research was to determine the extent of NA degradation by sediment microbial communities exposed to varying amounts of OSPW. METHODS AND RESULTS: Eleven wetlands, both natural and process-affected, and one tailings settling pond in Northern Alberta were studied. The natural wetlands and process-affected sites fell into two distinct groups based on their water chemistry. The extent of degradation of a 14C-labelled monocyclic NA surrogate [14C-cyclohexane carboxylic acid (CCA)] was relatively uniform in all sediments (approximately 30%) after 14 days. In contrast, degradation of a bicyclic NA surrogate [14C-decahydronaphthoic acid (DHNA)] was significantly lower in non process-affected sediments. Enrichment cultures, obtained from an active tailings settling pond, using commercially available NAs as the sole carbon source, resulted in the isolation of a co-culture containing Pseudomonas putida and Pseudomonas fluorescens. Quantitative GC-MS analysis showed that the co-culture removed >95% of the commercial NAs, and partially degraded the process NAs from OSPW with a resulting NA profile similar to that from 'aged wetlands'. CONCLUSIONS: Exposure to NAs induced and/or selected micro-organisms capable of more effectively degrading bicyclic NAs. Native Pseudomonas spp. extensively degraded fresh, commercial NA. The recalcitrant NAs resembled those found in process-affected wetlands. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that it may be possible to manipulate the existing environmental conditions to select for a microbial community exhibiting higher rates of NA degradation. This will have significant impact on the design of artificial wetlands for water treatment.     

Sperm flagellar motion maintained by ADP

Bull and human sperm which are dissected or impaled retain wave motion in the presence of 10 mM ADP. Unlike ATP-induced motility which must be mechanically initiated, ADP-induced motility requires no initiation. The activity observed appears similar to that of intact cells. Flagellar segments which are detached from the head and middle piece are capable of sustaining coordinated motility. Asymmetric motion resembling a ciliary beat can also be exhibited by free flagellar segments.     

Preservation of micro-organisms by drying; a review

The preservation of micro-organisms by different drying methodologies has been used for decades. Freeze drying in particular is the preferred method for transporting and storing vast culture collections of micro-organism strain types. The literature on drying and preserving micro-organisms is extensive, but is often specific to one particular strain. This review attempts to draw some similar concepts and findings together in one paper, to compare different drying techniques, with specific reference to microorganisms. The main topics covered are cell growth phases and concentration, inducing drying tolerance in microbial cells, drying methods, rehydration of dried cells and packaging and storage conditions. Also, particular attention has been paid to the use of freeze drying and the protective matrices used to improve microbial cell viability after drying.     

Ethylene production by micro-organisms grown on phenolic acids

In a 2-yr study, predators were shown to inflict heavy mortality on the eggs of codling moth, Cydia pomonella, placed in a cider-apple orchard where no insecticidal sprays were applied. Predatory Heteroptera sucked out 35 and 57 % of the eggs in the 2 yr respectively, and smaller numbers of eggs (16 %) disappeared, probably consumed by earwigs, Forficula auricularia. The potential of these predators for an integrated control system is briefly discussed.