Saturation mutagenesis of 2,4-DNT dioxygenase of Burkholderia sp. strain DNT for enhanced dinitrotoluene degradation

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon I204 of the alpha subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 +/- 0.03 and 0.22 +/- 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 +/- 0.6 nmol/min/mg protein and 4.7 +/- 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.     

Hydrogen bonding in diruthenium(II,III) tetraacetate complexes with biologically relevant axial ligands

Three diadduct complexes of the mixed-valent form of diruthenium tetraacetate, [Ru₂(μ-O₂CCH₃)₄L₂](PF₆), where L are the biologically relevant ligands imidazole, 1, 7-azaindole, 2, and caffeine, 3, were synthesized and characterized by elemental analysis, IR and UV-Vis spectroscopy and X-ray crystallography. In order to further elucidate the potential interactions of these dimers with DNA, the nature of the ligand coordination and the secondary inter- and intramolecular hydrogen-bonding interactions in all three complexes were assessed. Complex 1 · CH₂Cl₂ shows, exclusively, intermolecular interactions with the counterion whereas complexes 2 · ClCH₂CH₂Cl and 3 · OC(CH₃)₂ · H₂O, in addition to extensive intermolecular interactions, show intramolecular hydrogen bonding from the axial ligand to the bridging acetate oxygens, locking the ligand mean planes in place between the bridging acetate mean planes. In addition, all three complexes display π-π stacking of axial ligand rings on adjacent diadduct units.     

Distinct protein classes including novel merozoite surface antigens in Raft-like membranes of Plasmodium falciparum

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.     

Marine-derived nitrogen and carbon in freshwater-riparian food webs of the Copper River Delta,southcentral Alaska

After rearing to adulthood at sea, coho salmon (Oncorhynchus kisutch) return to freshwater to spawn once and then die on or near their spawning grounds. We tested the hypothesis that spawning coho salmon return marine N and C to beaver (Castor canadensis) ponds of the Copper River Delta (CRD), Cordova, southcentral Alaska, thereby enhancing productivity of the aquatic food webs that support juvenile coho salmon. We sampled three types of pond treatment: (1) natural enrichment by spawning salmon, (2) artificial enrichment via addition of salmon carcasses and eggs, and (3) ponds with no salmon enrichment. All ponds supported juvenile coho salmon. Seasonal samples of stable isotopes revealed that juvenile coho salmon, threespine sticklebacks (Gasterosteus aculeatus), caddisfly larvae, leeches, and chironomid midge larvae were enriched with marine N and C. The aquatic vascular plants bur reed (Sparganium hyperboreum), pondweed (Potamogeton gramineus), and mare’s tail (Hippuris vulgaris) were enriched with marine N only. Riparian vegetation (Sitka alder Alnus viridis ssp. sinuata and willow Salix spp.) did not show enrichment. Artificial additions of adult carcasses and eggs of coho salmon increased the δ¹⁵N and δ¹³C values of juvenile coho salmon. In this dynamic and hydrologically complex coastal environment, spawning coho salmon contributed marine N and C comprising 10–50% of the dietary needs of juvenile coho salmon through direct consumption of eggs and carcass material. Invertebrates that have assimilated marine N and C yield a further indirect contribution. This perennial subsidy maintains the productivity of the ecosystem of the coho salmon on the CRD.     

The diversity of Rab GTPases in Entamoeba histolytica

Rab proteins are ubiquitous small GTP-binding proteins that form a highly conserved family and regulate vesicular trafficking. Recent completion of the genome of the enteric protozoan parasite Entamoeba histolytica enabled us to identify an extremely large number (>90) of putative Rab genes. Multiple alignment and phylogenic analysis of amebic, human, and yeast Rab showed that only 22 amebic Rab proteins including EhRab1, EhRab2, EhRab5, EhRab7, EhRab8, EhRab11, and EhRab21 showed significant similarity to Rab from other organisms. The 69 remaining amebic Rab proteins showed only moderate similarity (<40% identity) to Rab proteins from other organisms. Approximately one-third of Rab proteins including Rab7, Rab11, and RabC form 15 subfamilies, which contain up to nine isoforms. Approximately 70% of amebic Rab genes contain single or multiple introns, and this proportion is significantly higher than that of common genes in this organism. Twenty-five Rabs possess an atypical carboxyl terminus such as CXXX, XCXX, XXCX, XXXC, and no cysteine. We propose annotation of amebic Rab genes and discuss biological significance of this extraordinary diversity of EhRab proteins in this organism.     

Balancing Akt with S6K: implications for both metabolic diseases and tumorigenesis

Proper regulation of the phosphoinositide 3-kinase-Akt pathway is critical for the prevention of both insulin resistance and tumorigenesis. Many recent studies have characterized a negative feedback loop in which components of one downstream branch of this pathway, composed of the mammalian target of rapamycin and ribosomal S6 kinase, block further activation of the pathway through inhibition of insulin receptor substrate function. These findings form a novel basis for improved understanding of the pathophysiology of metabolic diseases (e.g., diabetes and obesity), tumor syndromes (e.g., tuberous sclerosis complex and Peutz-Jegher's syndrome), and human cancers.     

Mechanical properties of calcified exoskeleton from the neotropical millipede, Nyssodesmus python

The calcified exoskeleton of millipedes plays a crucial role in resisting large forces developed during burrowing locomotion. I measured morphological and mechanical properties of cuticle from the neotropical forest floor millipede, Nyssodesmus python (Diplopoda: Polydesmidae), which ranges in body mass from 2 to 7 g. Scaling of thickness of the cuticle with respect to body mass followed predictions of geometric similarity. Both fracture strength and Young's modulus increased with body mass in females but not in males. In spite of their smaller size, male millipedes were still stronger, on average, than female millipedes. Mean fracture strength of millipede cuticle was 124 MPa, and Young's modulus was 17 GPa. Both of these values exceed measurements from typical insect cuticle, suggesting that calcium salts may play a role in stiffening and strengthening the millipede exoskeleton. Because of the high density of calcified millipede cuticle (1660 kg/m³), stiffness and strength relative to body weight remain comparable to values for other insect cuticles. These results corroborate a previous hypothesis that absolute not specific strength and stiffness have been selective factors in the evolution of millipede cuticle, and that bulkiness of the exoskeleton has been minimized through the deposition of calcium salts.     

Formation and composition of the Bacillus anthracis endospore

The endospores of Bacillus anthracis are the infectious particles of anthrax. Spores are dormant bacterial morphotypes able to withstand harsh environments for decades, which contributes to their ability to be formulated and dispersed as a biological weapon. We monitored gene expression in B. anthracis during growth and sporulation using full genome DNA microarrays and matched the results against a comprehensive analysis of the mature anthrax spore proteome. A large portion (approximately 36%) of the B. anthracis genome is regulated in a growth phase-dependent manner, and this regulation is marked by five distinct waves of gene expression as cells proceed from exponential growth through sporulation. The identities of more than 750 proteins present in the spore were determined by multidimensional chromatography and tandem mass spectrometry. Comparison of data sets revealed that while the genes responsible for assembly and maturation of the spore are tightly regulated in discrete stages, many of the components ultimately found in the spore are expressed throughout and even before sporulation, suggesting that gene expression during sporulation may be mainly related to the physical construction of the spore, rather than synthesis of eventual spore content. The spore also contains an assortment of specialized, but not obviously related, metabolic and protective proteins. These findings contribute to our understanding of spore formation and function and will be useful in the detection, prevention, and early treatment of anthrax. This study also highlights the complementary nature of genomic and proteomic analyses and the benefits of combining these approaches in a single study.     

A common cross-species function for the double epidermal growth factor-like modules of the highly divergent plasmodium surface proteins MSP-1 and MSP-8

An understanding of structural and functional constraints on the C-terminal double epidermal growth factor (EGF)-like modules of merozoite surface protein (MSP)-1 and related proteins is of importance to the development of these molecules as malaria vaccines and drug targets. Using allelic replacement, we show that Plasmodium falciparum parasites can invade erythrocytes and grow efficiently in the absence of an MSP-1 protein with authentic MSP-1 EGF domains. In this mutant parasite line, the MSP-1 EGFs were replaced by the corresponding double EGF module from P. berghei MSP-8, the sequence of which shares only low identity with its MSP-1 counterpart. Hence, the C-terminal EGF domains of at least some Plasmodium surface proteins appear to perform the same function in asexual blood-stage development. Mapping the surface location of the few residues that are common to these functionally complementary EGF modules revealed the presence of a highly conserved pocket of potential functional significance. In contrast to MSP-8, an even more divergent double EGF module, that from the sexual stage protein PbS25, was not capable of complementing MSP-1 EGF function. More surprisingly, two chimeric double EGF modules comprising hybrids of the EGF domains from P. falciparum and P. chabaudi MSP-1 were also not capable of replacing the P. falciparum MSP-1 EGF module. Together, these data suggest that although the MSP-1 EGFs can accommodate extensive sequence diversity, there appear to be constraints that may restrict the simple accumulation of point mutations in the face of immune pressure in the field.     

Identification of an Na(+)-dependent transporter associated with saxitoxin-producing strains of the cyanobacterium Anabaena circinalis

Blooms of the freshwater cyanobacterium Anabaena circinalis are recognized as an important health risk worldwide due to the production of a range of toxins such as saxitoxin (STX) and its derivatives. In this study we used HIP1 octameric-palindrome repeated-sequence PCR to compare the genomic structure of phylogenetically similar Australian isolates of A. circinalis. STX-producing and nontoxic cyanobacterial strains showed different HIP1 (highly iterated octameric palindrome 1) DNA patterns, and characteristic interrepeat amplicons for each group were identified. Suppression subtractive hybridization (SSH) was performed using HIP1 PCR-generated libraries to further identify toxic-strain-specific genes. An STX-producing strain and a nontoxic strain of A. circinalis were chosen as testers in two distinct experiments. The two categories of SSH putative tester-specific sequences were characterized by different families of encoded proteins that may be representative of the differences in metabolism between STX-producing and nontoxic A. circinalis strains. DNA-microarray hybridization and genomic screening revealed a toxic-strain-specific HIP1 fragment coding for a putative Na(+)-dependent transporter. Analysis of this gene demonstrated analogy to the mrpF gene of Bacillus subtilis, whose encoded protein is involved in Na(+)-specific pH homeostasis. The application of this gene as a molecular probe in laboratory and environmental screening for STX-producing A. circinalis strains was demonstrated. The possible role of this putative Na(+)-dependent transporter in the toxic cyanobacterial phenotype is also discussed, in light of recent physiological studies of STX-producing cyanobacteria.