Nitrogen-15 and Oxygen-18 Natural Abundance of Potassium Chloride Extractable Soil Nitrate Using the Denitrifier Method

In agroecosystems, most isotopic investigations of NO3- involve the use of tracers that are artificially enriched in 15N. Although the dual isotope composition of NO3-— d15N and d18O is especially beneficial for understanding the origin and fate of NO3-, its use for KCl-extractable soil NO3- has been hampered by the lack of a suitable analytical technique. Our objective was to test whether the denitrifier method, whereby NO3- is reduced to N2O before mass spectrometric analysis, can be used to determine the N and O isotopic composition of NO3- from 2 M KCl soil extracts. Several internationally accepted NO3- standards were dissolved in 2 M KCl, the conventional extractant for soil inorganic N, and inoculated with the bacterial strain Pseudomonas aureofaciens (ATCC no. 13985). The standard deviation of the NO3- standards analyzed did not exceed 0.2‰ for d15N and 0.3‰ for d18O values. After appropriate corrections, differences between our measured and consensus d15N and d18O values of standard NO3- generally were within the standard deviations given for the consensus values. Both d15N and d18O values were reproducible among separate analytical runs. The method was also tested on genuine 2 M KCl extracts from unfertilized and fertilized soils. Depending on N fertilization, the soils had distinct d15N and d18O values, which were attributed to amendment with NH4NO3 fertilizer. Hence, our data indicate that the denitrifier method provides a fast, reliable, precise, and accurate way of simultaneously analyzing the natural abundances of 15N and 18O in KCl-extractable soil NO3-.

A coiled-coil-repeat protein 'Ccrp' in Bdellovibrio bacteriovorus prevents cellular indentation, but is not essential for vibroid cell morphology

Bdellovibrio bacteriovorus are small, vibroid, predatory bacteria that grow within the periplasmic space of a host Gram-negative bacterium. The intermediate-filament (IF)-like protein crescentin is a member of a broad class of IF-like, coiled-coil-repeat-proteins (CCRPs), discovered in Caulobacter crescentus, where it contributes to the vibroid cell shape. The B. bacteriovorus genome has a single ccrp gene encoding a protein with an unusually long, stutter-free, coiled-coil prediction; the inactivation of this did not alter the vibriod cell shape, but caused cell deformations, visualized as chiselled insets or dents, near the cell poles and a general 'creased' appearance, under the negative staining preparation used for electron microscopy, but not in unstained, frozen, hydrated cells. Bdellovibrio bacteriovorus expressing 'teal' fluorescent protein (mTFP), as a C-terminal tag on the wild-type Ccrp protein, did not deform under negative staining, suggesting that the function was not impaired. Localization of fluorescent Ccrp-mTFP showed some bias to the cell poles, independent of the cytoskeleton, as demonstrated by the addition of the MreB-specific inhibitor A22. We suggest that the Ccrp protein in B. bacteriovorus contributes as an underlying scaffold, similar to that described for the CCRP protein FilP in Streptomyces coelicolor, preventing cellular indentation, but not contributing to the vibroid shape of the B. bacteriovorus cells.