Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

Laboratory investigations of mars: Chemical and spectroscopic characteristics of a suite of clays as Mars Soil Analogs

Two major questions have been raised by prior explorations of Mars. Has there ever been abundant water on Mars? Why is the iron found in the Martian soil not readily seen in the reflectance spectra of the surface? The work reported here describes a model soil system of Mars Soil Analog Materials, MarSAM, with attributes which could help resolve both of these dilemmas. The first set of MarSAM consisted of a suite of variably iron/calcium-exchanged montmorillonite clays. Several properties, including chemical composition, surface-ion composition, water adsorption isotherms, and reflectance spectra, of these clays have been examined. Also, simulations of the Viking Labeled Release Experiment using the MarSAM were performed. The results of these studies show that surface iron and adsorbed water are important determinants of clay behavior as evidenced by changes in reflectance, water absorption, and clay surface reactions. Thus, these materials provide a model soil system which reasonably satisfies the constraints imposed by the Viking analyses and remote spectral observations of the Martian surface, and which offers a sink for significant amounts of water. Finally, our initial results may provide insights into the mechanisms of reactions that occur on clay surfaces as well as a more specific approach to determining the mineralogy of Martian soils.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Clonal Analysis of Cell Division in the Bacillus subtilis div IV-B1 Minicell-Producing Mutant

Spores of the Bacillus subtilis minicell-producing mutant div IV-B1 were germinated and grown to microcolonies in chambers which facilitate continuous observation of the developing clones with a phase-contrast microscope. Time lapse photographs were taken of 46 clones, covering the period from the beginning of outgrowth until at least two rounds of cell division had been completed. Cell lineages were constructed from contour length measurements of the photographs. These data include cell lengths, division site locations, and cell numbers in clones of various ages. From these data we have determined that the probability of a minicell being produced at any division by the div IV-B1 mutant is 0.31. The location of the abnormal division site which generates the first minicell produced in the outgrowing clone appears to be random with respect to the existing cell poles. In contrast, the location of the second abnormal division site, and hence the second minicell, is not random but rather occurs preferentially in proximity to the first minicell. This clustering of abnormal events suggests that division site location is related to pole age (generations), although other influences on minicell clustering cannot be ruled out at present.     

Evolutionary Conservation of Sequences Directing Chromosome Breakage and Rdna Palindrome Formation in Tetrahymenine Ciliates

Extensive, programmed chromosome breakage occurs during formation of the somatic macronucleus of ciliated protozoa. The cis-acting signal directing breakage has been most rigorously defined in Tetrahymena thermophila, where it consists of a 15-bp DNA sequence known as Cbs, for chromosome breakage sequence. We have identified sequences identical or nearly identical to the T. thermophila Cbs at sites of breakage flanking the germline micronuclear rDNA locus of six additional species of Tetrahymena as well as members of two related genera. Other general features of the breakage site are also conserved, but surprisingly, the orientation and number of copies of Cbs are not always conserved, suggesting the occurrence of germline rearrangement events over evolutionary time. At one end of the T. thermophila micronuclear rDNA locus, a pair of short inverted repeats adjacent to Cbs directs the formation of a giant palindromic molecule. We have examined the corresponding sequences from two other Tetrahymena species. We find the sequence to be partially conserved, as previously implied from analysis of macronuclear rDNA, but of variable length and organization.     

Biomass production in a nitrogen-fertilized,tallgrass prairie ecosystem exposed to ambient and elevated levels of CO2

Increased biomass production in terrestrial ecosystems with elevated atmospheric CO₂ may be constrained by nutrient limitations as a result of increased requirement or reduced availability caused by reduced turnover rates of nutrients. To determine the short-term impact of nitrogen (N) fertilization on plant biomass production under elevated CO₂, we compared the response of N-fertilized tallgrass prairie at ambient and twice-ambient CO₂ levels over a 2-year period. Native tallgrass prairie plots (4.5 m diameter) were exposed continuously (24 h) to ambient and twice-ambient CO₂ from 1 April to 26 October. We compared our results to an unfertilized companion experiment on the same research site. Above- and belowground biomass production and leaf area of fertilized plots were greater with elevated than ambient CO₂ in both years. The increase in biomass at high CO₂ occurred mainly aboveground in 1991, a dry year, and belowground in 1990, a wet year. Nitrogen concentration was lower in plants exposed to elevated CO₂, but total standing crop N was greater at high CO₂. Increased root biomass under elevated CO₂ apparently increased N uptake. The biomass production response to elevated CO₂ was much greater on N-fertilized than unfertilized prairie, particularly in the dry year. We conclude that biomass production response to elevated CO₂ was suppressed by N limitation in years with below-normal precipitation. Reduced N concentration in above- and belowground biomass could slow microbial degradation of soil organic matter and surface litter, thereby exacerbating N limitation in the long term.     

The rfb locus from Pseudomonas aeruginosa strain PA103 promotes the expression of O antigen by both LPS-rough and LPS-smooth isolates from cystic fibrosis patients

Summary
Strains of Pseudomonas aeruginosa initially isolated from patients with cystic fibrosis (CF) often express a smooth lipopolysaccharide (LPS) containing many long O side-chain antigens, but once a chronic infection is established, strains recovered from these patients express little or no LPS O antigen. The genetic basis for this loss of O antigen expression by P. aeruginosa CF isolates is unknown. We report here that 20 CF isoiates of P. aeruginosa , 13 of which are LPS-rough, were each capable of expressing serogroup 011 antigen when provided with the rfb iocus from P. aeruginosa serogroup 011 strain PA103 on the recombinant plasmid pLPS2. Eight of the thirteen LPS-rough isolates co-expressed another, presumably endogenous, O antigen when they contained pLPS2. Different subcloned regions of pLPS2 complemented distinct strains to restore endogenous O antigen expression. These data suggest that the loss of O antigen expression by P. aeruginosa CF isolates results from alterations specific to the rfb region, and is not due to mutations involving other loci or ancillary LPS genes.     

THE GENETICS OF AN ISOLATING MECHANISM BETWEEN TWO SIBLING SPECIES OF DROSOPHILA

Matings between Drosophila simulans females and males of the sibling species D. mauritiana are of abnormally short duration. These rapid matings interrupt the transfer of sperm, leading to substantial reproductive isolation in interspecific as compared to intraspecific copulations. Genetic analysis of this behavior shows that it is influenced much more by the male than the female genotype, with genes from D. simulans being dominant. In males, shortened copulation is caused by interspecific divergence at a minimum of three loci, with one gene on each of the major chromosomes. This is an underestimate of the true number of loci affecting the trait, which could be much larger. The two autosomes have the largest effect, whereas that of the X chromosome is much smaller. The genetic architecture of copulation duration and the larger effect of male than female genotype suggest that females can detect and discriminate against differences in male genitalia.