Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Construction of Aspergillus nidulans strains producing enzymes of potential use in enology

Two different Aspergillus nidulans recombinant strains producing either the Aspergillus nidulans a-L-arabinofuranosidase A or a Candida molischiana b-glucosidase have been constructed. Depending on the growing conditions, the modified strains produce up to 4 or 18 times more b-glucosidase or a-L-arabinofuranosidase activity levels, respectively, than the wild type strain.     

Field Evaluation of Steinernema feltiae Against the Web-spinning Larch Sawfly Cephalcia lariciphila

Field trials were conducted in Rheola Forest, Wales, Great Britain, to determine the effectiveness of Steinernema feltiae UK strain in controlling the web-spinning larch sawfly Cephalcia lariciphila. Foliar sprays at the rate of 5,000-20,000 nematodes/100 cm branch resulted in 3.4-29.4% infection of sawfly larvae. Soil application of 200 nematodes/cm² resulted in 61% infection of sawfly prepupae and 17.3% of pupae. Prepupal infection ranged from 4.8 to 14.7% 1 year after nematode application. Soil applications of this nematode show that it has potential for biological control of sawfly prepupae.     

Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

Occurrence of a capsule in Aeromonas salmonicida

Aeromonas salmonicida grown in a medium with excess glucose as carbon source produces both capsular and exocellular polysaccharides. The capsular polysaccharide is composed of glucose, mannose, rhamnose, N-acetylmannosamine and mannuronic acid in the molar ratios of approximately 5:3:0.75:2:1. The extracellular polysaccharide is similarly constituted, but in the molar ratios of approximately 4.75:10.5:1.5:2:1. The capsular and exocellular polysaccharides did not cross-react with monoclonal antibodies against the A-layer or the O-antigen lipopolysaccharide.     

General recombination mechanisms in extracts of meiotic cells

RecA-like proteins have been purified from somatic and meiotic cells of mouse and lily. The rec proteins have been designated s-rec and m-rec to indicate their respective tissues of origin. The two proteins differ in molecular weight and in their response to temperature, the latter being consistent with the optimal temperature for physiological function of their tissues of origin. There is a major increase in m-rec protein with the entry of cells into meiosis, the peak of activity being early pachytene. Extracts of the cells and also those of yeast (Saccharomyces cerevisiae) have been prepared that have the capacity to catalyze homologous recombination. These extracts behave similarly to the m-rec proteins upon entry of cells into meiosis. Yeast transferred to sporulation medium displays a 100-fold increase in the recombination activity of the extract at about the time of entry into meiosis. The occurrence of peak levels of m-rec and recombination activity in extracts from cells in early pachytene points strongly to that stage as the time at which the enzymatic phase of recombination occurs.     

Value of Prospective Tissue Typing in Kidney Transplantation Between HL-A Identical Siblings

The predictability of leucocyte typing in kidney transplantation was assessed by an analysis of 37 kidney transplants from sibling donors. Recipients who were identical for the HL-A antigens with their donors gave highly predictable results. In comparison with those siblings who were incompatible or compatible but not identical their grafts functioned earlier, they required less immunosuppression, and had never had any rejections. They also appeared to have less postoperative morbidity. These results indicate that less immunosuppression than is current in many transplant centres could well be used with benefit in HL-A identical sibling transplants. This could reduce the risk of infection and possibly minimize the adverse effects of steroids on wound healing in these patients.     

Surface Antigens of Smooth Brucellae

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily.     

Evaluation of a method to measure conjugal transfer of recombinant DNA in soil slurries

Release of recombinant microbes into the environment necessitates an evaluation of their ability to transfer genetic material. The present report evaluates a method to detect conjugal DNA plasmid transfer in soil slurries under various environmental conditions. DonorPseudomonas cepacia containing pR388::Tn1721 andP. cepacia recipient cultures were coincubated in soil slurries containing autoclaved or natural soil and treated with one or more of 14 experimental conditions. Conjugal mating frequency (transconjugants per initial donor) ranged from 4.8×10^–1 to 1.9×10^–7. Highest numbers of transconjugants, 1.5×10⁷ colony forming units/ml soil slurry, were observed following incubation at 35°C with an enriched nutrient supplement added to the soil. Low numbers of transconjugants, 10³ colony forming units/ml soil slurry, were observed when mating pairs were subjected to low nutrient or pH stress even though initial donor and recipient populations were maintained at high levels. This test system provides a simple way to estimate effects of changing environmental factors on plasmid transfer rates and on the survival of recombinant microorganisms. By use of soil collected from sites proposed to receive genetically engineered microorganisms, preliminary risk assessments can be obtained regarding the potential for gene transfer and microorganism survival with this soil slurry test system.