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.     

Proteolytic Cleavage of Subunits of the Nucleocapsid of the Paramyxovirus Simian Virus 5

The protein subunits of the nucleocapsid of the parainfluenza virus simian virus 5 isolated from infected cells after dispersion with trypsin, chymotrypsin, or ficin are cleaved proteolytically. The molecular weights of the subunits which result from cleavage depend on the enzyme used, but are around 43,000, compared to the native subunit of 61,000. In most instances cleavage of the subunit appears to be due to the protease used to disperse the cell, and follows cell disruption. Nucleocapsids composed of native, uncleaved subunits can frequently be obtained from infected cells dispersed without a proteolytic enzyme; however, cleavage occasionally occurs even under those conditions, indicating that cellular proteases can at times cleave this protein. Nucleocapsids containing uncleaved subunits can be isolated from cells persistently infected with simian virus 5, indicating that persistent infection is not invariably associated with intracellular cleavage of this protein. Nucleocapsids composed of native subunits are hydrophobic, whereas those composed of the cleaved subunit can be dispersed in aqueous solution. It is suggested that the portion of the molecule removed by cleavage may be responsible for a specific interaction during virus assembly between the nucleocapsid and those areas of plasma membrane which contain the non-glycosylated viral membrane protein, which is also hydrophobic. An amino acid analysis of native and cleaved subunits has been done. The portion of the subunit removed by cleavage does not have a high proportion of hydrophobic residues, suggesting that those present are arranged together to form a hydrophobic domain.The N termini of both the native and cleaved subunits are blocked. This suggests that the portion of the molecule which is externally disposed and removed by cleavage contains the C terminus, and the cleaved subunit which reacts with the viral RNA contains the N terminus.     

Preparation of Agglutinating Antisera and Fluorescent-Antibody Conjugates Against Pasteurella tularensis in Equines

The serological response in burros and horses to the viable LVS strain of Pasteurella tularensis was studied. High-titered agglutinating antisera and fluorescent-antibody conjugates were obtained in both groups of animals. Maximum titers were obtained in horses 14 to 21 days after the start of vaccination and in burros 21 to 28 days after the start of vaccination. The use of Woodhour's adjuvants or booster inoculations did not result in increased titers.     

Proton nmr spectroscopy of high-spin ferrous cytochrome P-450 models

Alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins have been characterized in DMSO solution by proton nmr spectroscopy. A single mercaptide ligand binds to form a high-spin iron(II) complex as determined by solution magnetic measurements and the nmr isotropic shift pattern. Ligand exchange is slow on the nmr time scale unlike corresponding 2-methyl imidazole exchange rates which are very rapid. Further comparison of mercaptide and 2-methyl imidazole adducts reveals a downfield bias in isotropic shift values for the mercaptide species, which may be explained by different signs in the dipolar shift term for the two complexes. This apparent magnetic anisotropy of the mercaptide complex is in the same direction, although smaller, than that observed for bacterial cytochrome P-450. Isotropic shift values of at least 250 ppm for methylene resonances of the coordinated mercaptide support a very efficient unpaired spin delocalization for this axial ligand.