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.     

Effect of Cuticle Treatments on Infection of Phaseolus vulgaris by Rhizoctonia solani

Rhizoctonia solani, the causal agent of stem canker of beans, does not commonly infect beans more than two weeks old. Treatment of the cuticular layer by abrasion or chloroform rinsing results in decreased cuticular autofluorescence and the ability of stains to penetrate the hypocotyl surface. Infection cushions preferentially formed near these treated areas on both young and old plants. The morphology of infection cushions on older plants is simpler than those found on young, susceptible plants, but they are fully competent to form lesions.     

Identification of an outer mitochondrial membrane protein that interacts with a synthetic signal peptide

Chemical cross-linking procedures have been employed to study possible interactions between components of the mitochondrial outer membrane and NH2-terminal signal sequences located in proteins destined for import into the organelle. A synthetic peptide comprising amino acids 1-27 of pre-ornithine carbamyltransferase (pOCT) was found to interact specifically with a mitochondrial polypeptide of apparent molecular size 30 kDa. Membrane fractionation and protease accessibility analyses indicated that the polypeptide, designated p30, is located in the outer membrane. Binding of the synthetic peptide to p30 was saturable and reversible; Scatchard analysis of the binding data revealed a dissociation constant of 2 X 10(-6) M and predicts that p30 constitutes 4-10% of the outer mitochondrial membrane protein. Mild trypsin digestion of the mitochondrial surface destroyed both the ability of p30 to cross-link to the signal peptide and the ability of the organelle to import pOCT. Neither parameter was affected, however, by pretreatment of mitochondria with 1 M KCl.     

Progesterone levels and litter performance of sows following postpartum administration of prostaglandin F(2alpha)

A total of 101 sows was used to examine postpartum progesterone levels and litter performance following administration of 15 mg prostaglandins F(2alpha) (PGF(2alpha) n = 48) given within 12 h after farrowing. Daily blood samples and rectal temperatures were taken from all sows during the first 3 d post partum. Plasma progesterone (P(4)) concentrations were determined by radioimmunoassay (RIA). Regardless of treatment, plasma P(4) levels for all sows decreased in a similar fashion over the 3 d sampled. Mean (+/- SEM) P(4) on Day 2 (0.55 +/- 0.06 ng/ml) and Day 3 (0.38 +/- 0.04 ng/ml) were lower (P<0.01) than on Day 1 (0.98 +/- 0.08 ng/ml). Rectal temperature did not differ between PGF(2alpha) treated and nontreated sows nor was it different over the days measured. Litter characteristics, including survival rates on Day 7 and at weaning, and body weight on Days 3 and 35, were not affected by treatment. It was concluded that PGF(2alpha) administration to sows within 12 h post farrowing had no affect on the rate of luteal regression, as determined by P(4) concentration, nor on subsequent litter performance.     

Characterization of a cytoskeletal matrix associated with myelin from rat brain.

Extraction of rat brain myelin in a buffer containing Triton X-100 yielded a soluble fraction and an insoluble residue that was enriched in cytoskeletal elements. Immunoblot analysis of the detergent-soluble fraction and the insoluble cytoskeletal residue showed that all of the tubulin and more than half of the actin were found within the cytoskeletal fraction. The distribution of myelin-specific proteins was also examined, and revealed that 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) I and most of the myelin basic proteins (MBPs) were equally distributed between both fractions. By contrast, the large MBP (21.5 kDa) and CNPase II (50 kDa) were observed to partition almost entirely with the cytoskeletal fraction. Proteolipid protein was found predominantly in the detergent-soluble fraction, as was DM-20 protein. Analysis of the cytoskeletal fraction by sucrose-density-gradient centrifugation demonstrated that a distinct subset of lipids was tightly bound to the cytoskeletal protein residue. The cytoskeleton-associated lipid was considerably enriched in cerebroside and sphingomyelin by comparison with total myelin lipids. These results indicate that a cytoskeletal matrix is associated with multilamellar myelin, and suggest that this structure may play a fundamental role in myelinogenesis.     

Reaction of bovine-liver copper-zinc superoxide dismutase with hydrogen peroxide. Evidence for reaction with H2O2 and HO2- leading to loss of copper

The reaction of hydrogen peroxide with the copper-zinc bovine-liver superoxide dismutase at low molar ratios (0.2-20.0) of H2O2/active site between pH 7.3-10.0 leads to the loss of native enzyme as a distinct form monitored by electrophoresis. The pH dependence of the loss of native enzyme between 7.3 and 9.0 indicates the involvement of a conjugate base on the enzyme of pKa of 8.7 +/- 0.1. The rate of loss of the native enzyme is first order with respect to the concentration of both enzyme and hydrogen peroxide between pH 7.3 and 9.0 with no evidence for binding of peroxide. A second-order rate constant of 3.0 +/- 1.0 M-1 s-1 is obtained from these data. At pH 10.0 the reaction is first order with respect to enzyme concentration but saturable in H2O2. All data are consistent with the interpretation that H2O2 reacts with the enzyme at the lower pH where the reaction is dependent upon the conjugate base of a functional group on the enzyme. At the higher pH, the data are consistent with the reaction of HO2- and H2O2 with the dismutase. The dissociation constant for HO2- calculated from the kinetic data at pH 10.0 is between 25-50 microM and the rate constant for the breakdown of the HO2- dismutase complex is 1.10 + 0.05 x 10(-2) s-1. The change in the electrophoretic pattern at all pH values is accompanied by the loss of the ability of the enzyme to bind copper. Weakly bound or free copper can be detected using bathocuproine disulfonate. Furthermore copper-defficient forms of the enzyme can be detected by staining gels of the peroxide-treated dismutase with diethyldithiocarbamate.     

Structural relationships among chloramphenicol-resistance plasmids of Staphylococcus aureus

Abstract Twelve different chloramphenicol-resistance (Cm^r) plasmids detected in Staphylococcus aureus strains isolated between 1952 and 1981 were characterized by restriction endonuclease, DNA hybridization and heteroduplex analyses. These studies revealed three families of Cm^r plasmids which were distinguished by their chloramphenicol acetyltransferase sequences; the prototype plasmids of the families were pC221, pC223 and pC194. The cat and replication ( rep ) genes of the plasmid pC221 were highly conserved in other pC221 family members and were related to their homologs in the pC223 family plasmids; however, the cat and rep genes of the pC194 family plasmids were distinct.     

Gravitropism in Higher Plant Shoots : V. Changing Sensitivity to Auxin

An alternative to the Cholodny-Went, auxin-transport hypothesis of gravitropic stem bending was proposed as early as 1958, suggesting that gravistimulation induces changes in sensitivity to auxin, accounting for differential growth and bending. To test the sensitivity hypothesis, we immersed marked, decapitated sunflower (Helianthus annuus L.) hypocotyl sections in buffered auxin solutions over a wide concentration range (0, 10⁻⁸ to 10⁻² molar IAA), photographed them at half-hour intervals, analyzed the negatives with a digitizer/computer, and evaluated surface-length changes in terms of Michaelis-Menten enzyme kinetics. Bending decreases with increasing auxin concentration; above about 10⁻⁴ molar IAA the hypocotyls bend down; increasing auxin inhibits elongation growth of lower surfaces (which is high at zero or relatively low auxin levels) but promotes upper-surface growth (which is low at low auxin levels). Thus, lower surfaces have a greater K_m sensitivity to applied auxin than upper surfaces. At optimum auxin levels (maximum growth), growth of bottom surfaces exceeds that of top surfaces, so bottom tissues have a greater V_max sensitivity. V_max sensitivity of vertical controls is slightly lower than it is for either horizontal surface; K_m sensitivity is intermediate. Clearly, gravistimulation leads to significant changes in tissue sensitivity to applied auxin. Perhaps these changes are also important in normal gravitropism.     

Influence of oxygen tension on the respiratory activity of Mycobacterium phlei

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, beta-hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction.