Lateral Transfer of Genes for Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) Degradation

Recent studies demonstrated that degradation of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by species of Rhodococcus, Gordonia, and Williamsia is mediated by a novel cytochrome P450 with a fused flavodoxin reductase domain (XplA) in conjunction with a flavodoxin reductase (XplB). Pulse field gel analysis was used to localize xplA to extrachromosomal elements in a Rhodococcus sp. and distantly related Microbacterium sp. strain MA1. Comparison of Rhodococcus rhodochrous 11Y and Microbacterium plasmid sequences in the vicinity of xplB and xplA showed near identity (6,710 of 6,721 bp). Sequencing of the associated 52.2-kb region of the Microbacterium plasmid pMA1 revealed flanking insertion sequence elements and additional genes implicated in RDX uptake and degradation.Past practices of production, application, and disposal of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have resulted in widespread contamination. Environmental contamination is aggravated by its high mobility, contributing to more-widespread contamination of groundwater than by other commonly used explosives (27). Ingestion or inhalation of RDX is associated with neurological disorders and organ failure (38), and exposed wildlife show behavioral changes and suffer liver and reproductive damage (38). The U.S. Environmental Protection Agency (EPA) has classified RDX as a possible human carcinogen (37). These adverse effects have provided motivation to better understand the microbiology and biochemistry of RDX degradation.As yet there is relatively limited information concerning natural rates of microbial RDX degradation or degradation mechanisms; such information is needed to predict or control rates of degradation in the environment. Of the three general pathways for RDX degradation or transformation based on metabolite analysis outlined in the review by Crocker and associates (6), aerobic degradation initiated by XplA is among the better-characterized systems. This enzyme, a novel cytochrome P450 with a fused flavodoxin reductive domain (18, 33), was first identified by Seth-Smith et al. in Rhodococcus rhodochrous 11Y as being encoded by xplA (33). This gene has been identified in 24 bacterial isolates of the Corynebacterineae capable of utilizing RDX as a sole nitrogen source (4, 25, 32-34). While mammalian nitric oxide synthase family enzymes are known to be P450-like enzymes with fused flavodoxin domains, there are very few identified examples of this type of protein fusion among characterized microbial species (2, 15, 18, 24). Subsequent studies by Jackson and associates (18) demonstrated that XplA, in association with an electron-transferring flavodoxin reductase (XplB), functions to efficiently denitrate RDX aerobically to the aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB) (18). NDAB has been shown to serve as a viable nitrogen source for Methylobacterium sp. strain JS178 (13) and to be degraded by Phanerochaete chrysosporium (11). Thus, complete mineralization often appears to be mediated by multiple microbial populations.The capacity for microbial degradation of recalcitrant organics, many of which are apparently new to the biosphere as a result of chemical manufacture, is often determined by plasmids and associated mobile genetic elements (36, 39). Plasmids both serve as a reservoir of genetic information and promote metabolic innovation, since their replication is independent of the chromosome and they do not generally encode essential functions. Although it was earlier suggested that genes in Rhodococcus sp. strain DN22 associated with initial steps of RDX degradation are carried by plasmids (5), no direct evidence for an extrachromosomal location was provided. We now show that nearly identical genes for XplA and XplB are carried on plasmids in two phylogenetically and geographically distinct bacterial isolates: Microbacterium sp. strain MA1, isolated from North America (Milan, TN), and Rhodococcus rhodochrous 11Y, isolated from England (33). Thus, these genes are more broadly distributed within the Actinomycetales than previously recognized, and the near identity of gene sequence (6,710 of 6,721 bp) in these divergent genera is indicative of recent plasmid-mediated transfer. Analysis of approximately 52 kbp of sequence near xplA and xplB in strain MA1 revealed closely linked genes for transport and degradation that are flanked by transposable elements, suggesting that plasmid-carried xplA and xplB are part of a larger class I transposable element encoding both transport and degradation of RDX.     

Lumbricus erythrocruorin at 3.5 A resolution: architecture of a megadalton respiratory complex

Annelid erythrocruorins are highly cooperative extracellular respiratory proteins with molecular masses on the order of 3.6 million Daltons. We report here the 3.5 A crystal structure of erythrocruorin from the earthworm Lumbricus terrestris. This structure reveals details of symmetrical and quasi-symmetrical interactions that dictate the self-limited assembly of 144 hemoglobin and 36 linker subunits. The linker subunits assemble into a core complex with D(6) symmetry onto which 12 hemoglobin dodecamers bind to form the entire complex. Although the three unique linker subunits share structural similarity, their interactions with each other and the hemoglobin subunits display striking diversity. The observed diversity includes design features that have been incorporated into the linker subunits and may be critical for efficient assembly of large quantities of this complex respiratory protein.     

Genomic characterization of POS5, the Saccharomyces cerevisiae mitochondrial NADH kinase

Disruption of the Saccharomyces cerevisiae mitochondrial NADH kinase POS5 increases the mitochondrial mutation rate 50-fold. Whereas most multicellular eukaryotic genomes have one NADH kinase gene, the yeast genome contains three distinct genes encoding NAD/H kinase activity. To determine if all three genes are essential for viability we constructed combinations of gene knockouts. We show that only the pos5Deltautr1Delta combination is synthetically lethal, demonstrating an essential overlapping function, and showing that NAD/H kinase activity is essential for eukaryotic viability. The single human NAD/H kinase gene can rescue the lethality of the double knockout in yeast, demonstrating that the single human gene can fill the various functions provided by the three yeast genes. The human NAD/H kinase gene harbors very common sequence variants, but all of these equally complement the synthetic lethality in yeast, illustrating that each of these are functionally wild-type. To understand the molecular mechanism of the mitochondrial genome instability of pos5 mutation we performed gene expression analysis on the pos5Delta. The pos5Delta resulted in an increase in expression of most of the iron transport genes including key genes involved in iron-sulfur cluster assembly. Decreased expression occurred in many genes involved in the electron transport chain. We show that the pos5Delta expression pattern is similar to the frataxin homolog knockout (yfh1Delta), the yeast model for Friedreich's ataxia. These combined data show that the POS5 NAD/H kinase is an important protein required for a variety of essential cellular pathways and that deficient iron-sulfur cluster assembly may play a critical role in the mitochondrial mutator phenotype observed in the pos5Delta.     

Effect of light internsity on vesicle formation in Chlorobium

1. Chlorobium limicola forma sp. thiosulfatophilum was cultivated at 22 and 22000 lux. 2. The content of bchl d on a protein basis in the low light intensity cultures was about twice that of the high light intensity cultures. 3. After growth at 22 lux the red bchl d peak was at c. 743 nm, while at the higher intensity this peak was at c. 732 nm. 4. Electron microscopy of thin sections of Chlorobium revealed that vesicle size was greater at the low light intensity than at the high. 5. This was confirmed by sucrose density gradient centrifugation of differentially ¹⁴C-labelled vesicles from cultures grown at the two intensities. 6. The optimum temperature for growth was about 35°C. Incubation at the optimum temperature was particularly beneficial at high light intensity.Abbreviation bchl bacteriochlorophyll     

Crystal structural studies of changes in the native dinuclear iron center of ribonucleotide reductase protein R2 from mouse

Class I ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides in mammals and many other organisms. The RNR subunit R2 contains a dinuclear iron center, which in its diferrous form spontaneously reacts with O2, forming a mu-oxo-bridged diferric cluster and a stable tyrosyl radical. Here, we present the first crystal structures of R2 from mouse with its native dinuclear iron center, both under reducing and oxidizing conditions. In one structure obtained under reducing conditions, the iron-bridging ligand Glu-267 adopts the mu-(eta1,eta2) coordination mode, which has previously been related to O2 activation, and an acetate ion from the soaking solution is observed where O2 has been proposed to bind the iron. The structure of mouse R2 under oxidizing conditions resembles the nonradical diferric R2 from Escherichia coli, with the exception of the coordination of water and Asp-139 to Fe1. There are also additional water molecules near the tyrosyl radical site, as suggested by previous spectroscopic studies. Since no crystal structure of the active radical form has been reported, we propose models for the movement of waters and/or tyrosyl radical site when diferric R2 is oxidized to the radical form, in agreement with our previous ENDOR study. Compared with E. coli R2, two conserved phenylalanine residues in the hydrophobic environment around the diiron center have opposing rotameric conformations, and the carboxylate ligands of the diiron center in mouse R2 appear more flexible. Together, this might contribute to the lower affinity and cooperative binding of iron in mouse R2.     

Specific residues in plasmatocyte-spreading peptide are required for receptor binding and functional antagonism of insect immune cells

Plasmatocyte-spreading peptide (PSP) is a 23-amino acid cytokine that activates a class of insect immune cells called plasmatocytes. PSP consists of two regions: an unstructured N terminus (1-6) and a highly structured core (7-23). Prior studies identified specific residues in both the structured and unstructured regions required for biological activity. Most important for function were Arg13, Phe3, Cys7, Cys19, and the N-terminal amine of Glu1. Here we have built on these results by conducting cell binding and functional antagonism studies. Alanine replacement of Met12 (M12A) resulted in a peptide with biological activity indistinguishable from PSP. Competitive binding experiments using unlabeled and 125I-M12A generated an IC50 of 0.71 nm and indicated that unlabeled M12A, at concentrations > or =100 nm, completely blocked binding of label to hemocytes. We then tested the ability of other peptide mutants to displace 125I-M12A at a concentration of 100 nm. In the structured core, we found that Cys7 and Cys19 were essential for cell binding and functional antagonism, but these effects were likely because of the importance of these residues for maintaining the tertiary structure of PSP. Arg13, in contrast, was also essential for binding and activity but is not required for maintenance of structure. In the unstructured N-terminal region, deletion of the phenyl group from Phe3 yielded a peptide that reduced binding of 125I-M12A 326-fold. This and all other mutants of Phe3 we bioassayed were unable to antagonize PSP. Deletion of Glu1 in contrast had almost no effect on binding and was a strong functional antagonist. Experiments using a photoaffinity analog indicated that PSP binds to a single 190-kDa protein.     

Population structure, mating system, and sex-determining allele diversity of the parasitoid wasp Habrobracon hebetor

Besides haplo-diploid sex determination, where females develop from fertilized diploid eggs and males from unfertilized haploid eggs, some Hymenoptera have a secondary system called complementary sex determination (CSD). This depends on genotypes of a 'sex locus' with numerous sex-determining alleles. Diploid heterozygotes develop as females, but diploid homozygotes become sterile or nonviable diploid males. Thus, when females share sex-determining alleles with their mates and produce low fitness diploid males, CSD creates a genetic load. The parasitoid wasp Habrobracon hebetor has CSD and displays mating behaviours that lessen CSD load, including mating at aggregations of males and inbreeding avoidance by females. To examine the influence of population structure and the mating system on CSD load, we conducted genetic analyses of an H. hebetor population in Wisconsin. Given the frequency of diploid males, we estimated that the population harboured 10-16 sex-determining alleles. Overall, marker allele frequencies did not differ between subpopulations, but frequencies changed dramatically between years. This reduced estimates of effective size of subpopulations to only N3 approximately 20-50, which probably reflected annual fluctuations of abundance of H. hebetor. We also determined that the mating system is effectively monogamous. Models relating sex-determining allele diversity and the mating system to female productivity showed that inbreeding avoidance always decreased CSD loads, but multiple mating only reduced loads in populations with fewer than five sex-determining alleles. Populations with N3 less than 100 should have fewer sex-determining alleles than we found, but high diversity could be maintained by a combination of frequency-dependent selection and gene flow between populations.     

Genetic heterogeneity of stably transfected cell lines revealed by expression profiling with oligonucleotide microarrays

Large-scale gene expression measurements with oligonucleotide microarrays have contributed tremendously to biological research. However, to distinguish between relevant expression changes and falsely identified positives, the source and magnitude of errors must be understood. Here, we report a source of biological variability in microarray experiments with stably transfected cell lines. Mouse embryonic fibroblast (MEF/3T3) and rat schwannoma (RT4) cell lines were generated to provide regulatable schwannomin expression. The expression levels of 29 samples from five different mouse embryonic fibroblast clonal cell lines and 18 samples from 3 RT4 cell lines were monitored with oligonucleotide microarrays. Using hierarchical clustering, we determined that the changes in gene expression induced by schwannomin overexpression were subtle when compared with those detected as a consequence of clonal selection during generation of the cell lines. The hierarchical clustering implies that significant alterations of gene expression were introduced during the transfection and selection processes. A total of 28 genes were identified by Kruskal-Wallis rank test that showed significant variation between clonal lines. Most of them were related to cytoskeletal function and signaling pathways. Based on these analyses, we recommend that replications of experiments with several selected cell lines are necessary to assess biological effects of induced gene expression.     

A comparative analysis of the impact on biota and man in the 30 km zone of the Chernobyl NPP

A methodological approach is described for a comparative assessment of ionizing radiation effects on man and biota, based on the use of indices of radiation impact--ratios of actual exposure doses to environmental objects (including humans) and critical ones. As such doses, standards limiting radiation exposure and doses at which phenotypical effects were absent after the Chernobyl accident were employed, respectively for man and biota. For the test site chosen within the 30 km ChNPP zone (region of the Borshchovka settlement), dose burdens to reference biota species and the population (with and without evacuation) and the corresponding radiation impact indices were calculated. For the long term period after the accident radiation safety standards for man are shown to ensure radiation safety for biota as well. At the same time in the early period after the accident the emergency regulations do not guarantee adequate protection of nature, some species of which can be subject to irradiation more than man, even if countermeasures like evacuation are not applied. A conclusion has been made on the necessity of a more detailed and comprehensive analysis of situations when the anthropocentric principle "if radiation standards protect man then biota are also adequately protected" is violated.