γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined. The recent identification of a species-specific acceptor site inhibitor, OU749, suggests that these inhibitors may be less toxic than glutamine analogues. Here we investigated the donor and acceptor substrate preferences of Bacillus anthracis GGT (CapD) and applied computational approaches in combination with kinetics to probe the structural basis of the enzyme's substrate and inhibitor binding specificities and compare them with human GGT. Site-directed mutagenesis studies showed that the R432A and R520S variants exhibited 6- and 95-fold decreases in hydrolase activity, respectively, and that their activity was not stimulated by the addition of the l-Cys acceptor substrate, suggesting an additional role in acceptor binding and/or catalysis of transpeptidation. Rat GGT (and presumably HuGGT) has strict stereospecificity for L-amino acid acceptor substrates, while CapD can utilize both L- and D-acceptor substrates comparably. Modeling and kinetic analysis suggest that R520 and R432 allow two alternate acceptor substrate binding modes for L- and D-acceptors. R432 is conserved in Francisella tularensis, Yersinia pestis, Burkholderia mallei, Helicobacter pylori and Escherichia coli, but not in human GGT. Docking and MD simulations point toward key residues that contribute to inhibitor and acceptor substrate binding, providing a guide to designing novel and specific GGT inhibitors.

Bacillus anthracis, the causative agent of anthrax, is recognized as one of the most serious bioterrorism threats. The current human vaccines are based on the protective antigen component of the anthrax toxins. Concern about possible vaccine resistant strains and reliance on a single antigen has prompted the search for additional immunogens. Bacterial capsules, as surface-expressed virulence factors, are well-established components of several licensed vaccines. In a previous study we showed that an anthrax vaccine consisting of the B. anthracis poly-γ-d-glutamic acid capsule covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B protected mice against parenteral B. anthracis challenge. Here we tested this vaccine in rabbits and monkeys against an aerosol spore challenge. The vaccine induced anti-capsule antibody responses in both species, measured by ELISA and a macrophage opsono-adherence assay. While rabbits were not protected against a high aerosol challenge dose, significant protection was observed in monkeys receiving the capsule conjugate vaccine. The results confirm that the capsule is a protective immunogen against anthrax, being the first non-toxin antigen shown to be efficacious in monkeys and suggest that addition of capsule may broaden and enhance the protection afforded by protective antigen-based vaccines.

The Line Islands are calcium carbonate coral reef platforms located in iron-poor regions of the central Pacific. Natural terrestrial run-off of iron is non-existent and aerial deposition is extremely low. However, a number of ship groundings have occurred on these atolls. The reefs surrounding the shipwreck debris are characterized by high benthic cover of turf algae, macroalgae, cyanobacterial mats and corallimorphs, as well as particulate-laden, cloudy water. These sites also have very low coral and crustose coralline algal cover and are call black reefs because of the dark-colored benthic community and reduced clarity of the overlying water column. Here we use a combination of benthic surveys, chemistry, metagenomics and microcosms to investigate if and how shipwrecks initiate and maintain black reefs. Comparative surveys show that the live coral cover was reduced from 40 to 60% to <10% on black reefs on Millennium, Tabuaeran and Kingman. These three sites are relatively large (>0.75 km(2)). The phase shift occurs rapidly; the Kingman black reef formed within 3 years of the ship grounding. Iron concentrations in algae tissue from the Millennium black reef site were six times higher than in algae collected from reference sites. Metagenomic sequencing of the Millennium Atoll black reef-associated microbial community was enriched in iron-associated virulence genes and known pathogens. Microcosm experiments showed that corals were killed by black reef rubble through microbial activity. Together these results demonstrate that shipwrecks and their associated iron pose significant threats to coral reefs in iron-limited regions.

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.

Difficulties in scaling up theoretical and experimental results have raised controversy over the consequences of biodiversity loss for the functioning of natural ecosystems. Using a global survey of reef fish assemblages, we show that in contrast to previous theoretical and experimental studies, ecosystem functioning (as measured by standing biomass) scales in a non-saturating manner with biodiversity (as measured by species and functional richness) in this ecosystem. Our field study also shows a significant and negative interaction between human population density and biodiversity on ecosystem functioning (i.e., for the same human density there were larger reductions in standing biomass at more diverse reefs). Human effects were found to be related to fishing, coastal development, and land use stressors, and currently affect over 75% of the world's coral reefs. Our results indicate that the consequences of biodiversity loss in coral reefs have been considerably underestimated based on existing knowledge and that reef fish assemblages, particularly the most diverse, are greatly vulnerable to the expansion and intensity of anthropogenic stressors in coastal areas.

Blacktip reef sharks Carcharhinus melanopterus were the most abundant predator in the lagoons at Palmyra Atoll. They were evenly distributed throughout the lagoons, although there was some evidence of sexual segregation. Males reach sexual maturity between 940-1,020 mm L(T). Bird remains were found in some C. melanopterus stomachs. C. melanopterus at Palmyra appear to be smaller than those at other locations.

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have approximately 1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.

A series of surveys were carried out to characterize the physical and biological parameters of the Millennium Atoll lagoon during a research expedition in April of 2009. Millennium is a remote coral atoll in the Central Pacific belonging to the Republic of Kiribati, and a member of the Southern Line Islands chain. The atoll is among the few remaining coral reef ecosystems that are relatively pristine. The lagoon is highly enclosed, and was characterized by reticulate patch and line reefs throughout the center of the lagoon as well as perimeter reefs around the rim of the atoll. The depth reached a maximum of 33.3 m in the central region of the lagoon, and averaged between 8.8 and 13.7 m in most of the pools. The deepest areas were found to harbor large platforms of Favia matthaii, which presumably provided a base upon which the dominant corals (Acropora spp.) grew to form the reticulate reef structure. The benthic algal communities consisted mainly of crustose coralline algae (CCA), microfilamentous turf algae and isolated patches of Halimeda spp. and Caulerpa spp. Fish species richness in the lagoon was half of that observed on the adjacent fore reef. The lagoon is likely an important nursery habitat for a number of important fisheries species including the blacktip reef shark and Napoleon wrasse, which are heavily exploited elsewhere around the world but were common in the lagoon at Millennium. The lagoon also supports an abundance of giant clams (Tridacna maxima). Millennium lagoon provides an excellent reference of a relatively undisturbed coral atoll. As with most coral reefs around the world, the lagoon communities of Millennium may be threatened by climate change and associated warming, acidification and sea level rise, as well as sporadic local resource exploitation which is difficult to monitor and enforce because of the atoll's remote location. While the remote nature of Millennium has allowed it to remain one of the few nearly pristine coral reef ecosystems in the world, it is imperative that this ecosystem receives protection so that it may survive for future generations.

Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and alpha-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.