Macroalgal blooms on southeast Florida coral reefs: II. Cross-shelf discrimination of nitrogen sources indicates widespread assimilation of sewage nitrogen

Since 1990, coral reefs off southeast Florida have experienced an unprecedented succession of macroalgal blooms and invasions. To determine if anthropogenic land-based nitrogen (N) sources support these HABs, we collected macroalgal tissue for stable nitrogen isotope (δ¹⁵N) analysis at three spatially distinct depths ranging from the shallow subtidal to the shelf break (43 m) along seven transects from Jupiter to Deerfield Beach, Florida, USA. This sampling was initiated during a historically significant drought in the spring of 2001 when rainfall, stormwater runoff, and upwelling were at a minimum. The sampling was repeated in the summer wet season following significant stormwater runoff and during a strong upwelling event.Despite significant seasonal changes in N source availability, δ¹⁵N values did not vary between samplings. Collectively, δ¹⁵N values were significantly higher on inshore shallow reefs (+8.1‰) compared to mid (+6.1‰) and deep reefs (+6.7‰). Values were also elevated in the southern portion of the study area (e.g., Boca Raton, +8.5‰) where nearly 1.5 billion l/day of secondarily treated wastewater is discharged into the ocean via coastal outfalls. Codium isthmocladum, a macroalga that assimilates its nutrients entirely from the water column, was the dominant bloom species in the southern study area, where tissue δ¹⁵N values matched source values of nearby sewage outfalls. The northern study area was dominated by species of the genus Caulerpa, particularly the invasive Pacific native C. brachypus var. parvifolia, which are capable of accessing benthic nutrient sources (e.g., submarine groundwater discharge, SGD) by means of root-like rhizoids. The northern area does not have sewage outfalls but features a highly transmissive geologic zone where SGD may be enriched with septic tank leachate and effluent from municipal deep injection wells.Multiple lines of evidence supported the hypothesis that land-based sewage N was more important than upwelling as a N source to these HABs: (1) δ¹⁵N values were highest on shallow reefs and decreased with increasing depth, indicating land-based sources of enrichment; (2) elevated δ¹⁵N values occurred in these HABs during the dry season, prior to the onset of the summer upwelling; (3) elevated NH₄⁺ concentrations occur on these reefs during both upwelling and non-upwelling periods and are kinetically preferred by macroalgae compared to upwelled NO₃⁻. These findings provide a case study of a coupling between increasing anthropogenic activities and the development of macroalgal HABs, including invasive species that threaten economically important reef resources in southeast Florida.     

The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.     

Polo-box domains confer target specificity to the Polo-like kinase family

Polo-like kinases (Plks) contain a conserved Polo-box domain, shown to bind to phosphorylated Ser-pSer/pThr-Pro motifs. The Polo-box domain of Plk-1 mediates substrate interaction and plays an important role in subcellular localization. Intriguingly, the major interactions between the PBD and the optimal recognition peptide are mediated by highly conserved residues in the PBD, suggesting there is little target specificity conveyed by the various PBDs. However, here we show that the affinity of the purified Plk1-3 PBDs to both a physiological Cdc25C derived phospho-peptide and an optimal recognition phospho-peptide differs significantly among family members. To decipher the role of the PBDs and kinase domains in inferring Plk specificity, we exchanged the PBD of Plk1 (PBD1) with the PBD of Plk2, 3, or 4 (PBD2-4). The resulting hybrid proteins can restore bipolar spindle formation and centrosome maturation in Plk1-depleted U2OS cells to various degrees. In these experiments PBD2 was most efficient in complementing PBD-function. Using the MPM2 antibody that recognizes a large set of mitotic phospho-proteins, we could show that PBD1 and PBD2 display some limited overlap in target recognition. Thus, PBDs convey a significant deal of target specificity, indicating that there is only a limited amount of functional redundancy possible within the Plk family.     

Comparison of vertebrate and invertebrate CLIC proteins: the crystal structures of Caenorhabditis elegans EXC-4 and Drosophila melanogaster DmCLIC

The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid.     

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

Estimation of heterozygosity for single-probe multilocus DNA fingerprints

In spite of the increasing application of DNA fingerprinting to natural populations and to the genetic identification of humans, explicit methods for estimation of basic population genetic parameters from DNA fingerprinting data have not been developed. Contributing to this omission is the inability to determine, for multilocus fingerprinting probes, relatively important genetic information, such as the number of loci, the number of alleles, and the distribution of these alleles into specific loci. One of the most useful genetic parameters that could be derived from such data would be the average heterozygosity, which has traditionally been employed to measure the level of genetic variation within populations and to compare genetic variation among different loci. We derive here explicit formulas for both the estimation of average heterozygosity at multiple hypervariable loci and a maximum value for this estimate. These estimates are based upon the DNA restriction-pattern matrices that are typical for fingerprinting studies of humans and natural populations. For several empirical data sets from our laboratory, estimates of average and maximal heterozygosity are shown to be relatively close to each other. Furthermore, variances of these statistics based on simulation studies are relatively small. These observations, as well as consideration of the effect of missing alleles and alternate numbers of loci, suggest that the average heterozygosity can be accurately estimated using phenotypic DNA fingerprint patterns, because this parameter is relatively insensitive to the lack of certain genetic information.      

The Barrier Reef sediment apron: Tobacco Reef,Belize

Sedimentological and biological surveys of the back-reef sediment apron of Tobacco Reef, a continuous segment of the Belizean Barrier Reef, reveal five distinct biogeological zones: (1) coralline-coral-Dictyota pavement, (2) Turbinaria-Sargassum rubble, (3) Laurencia-Acanthophora sand and gravel, (4) bare sand and 95 Thalassia sand. These zones parallel the entire 9-km reef. The distribution of these zones is related to the spatial patterns of fish herbivory, the size of bottom sediments, and the stability of the substrate. Sedimentological and hydrodynamic studies indicate that most of the sediments in this area are transported from the reef crest and fore reef during periods of storm or hurricane activity and that their size distribution is largely the result of differential transport by high bottom-water velocities during those periods.     

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.