The dynamics of benthic microbial communities at Davies Reef,central Great Barrier Reef

The dynamics of benthic microbial communities were examined within different functional zones (reef crest, reef flat, lagoon) of Davies Reef, central Great Barrier Reef, in winter. Bacterial numbers did not change significantly across the reef with a mean abundance of 1.3 (±0.6) x 10⁹ cells g^-1 DW of sediment. Bacterial production, measured as thymidine incorporation into DNA, ranged from 1.2 (±0.2) to 11.6 (±1.5) mg C m^-2h^-1 across the reef and was significantly lower in a reef crest basin than in the other zones. Bacterial growth rates () across the reef (0.05 to 0.33 g^-1) correlated only with sediment organic carbon and nitrogen. Protozoan and meiofaunal densities varied by an order of magnitude across the reef and correlated with one or more sediment variables but not with bacterial numbers or growth rates. Nutrient flux rates were similar to those found at other reefs in the central and southern Great Barrier Reef and are significantly lower than rates measured in temperate sand communities. In the front lagoon, bioturbation and feeding acitivity by thalassinid shrimps (Callianassa spp.) negatively influenced microbial and meiofaunal communities with a net import of organic matter necessary to support the estimated rates of bacterial productivity. In lagoonal areas not colonized by shrimps, primary productivity (400–1100 mg C m^-2d^-1) from algal mats was sufficient to support bacterial growth. It is suggested that deposit-feeding macrobenthos such as thalassinid crustaceans play a major role in the tructuring and functioning of lower trophic groups (bacteria, microalgae, protozoa, meiofauna) in coral reef sedments, particularly in laggons.     

Preliminary evaluation of non-native rainbow trout (Oncorhynchus mykiss) impact on the Cederberg ghost frog (Heleophryne depressa) in South Africa’s Cape Fold Ecoregion

We evaluated the impact of non-native rainbow trout Oncorhynchus mykiss on a population of endemic Cedarberg ghost frog Heleophryne depressa in the upper Krom River (Olifants-Doring River Catchment, Cape Fold Ecoregion). We compared H. depressa abundance (using kick-sampling and underwater video analysis) and environmental conditions between sites above and below a waterfall that marks the upper distribution limit of O. mykiss. Heleophryne depressa abundance was significantly greater above the waterfall than that below it, and, because there was no significant difference in measured environmental variables, O. mykiss presence is identified as the most likely explanation for the observed decrease in H. depressa abundance.     

Absence of correlation between chemo- and radioresistance in a range of human tumour cell lines

The correlation between cellular resistance to radiation and to chemotherapeutic drugs has been investigated in a number of solid tumour cell lines, and preliminary results indicate no direct relationship. The acquisition of a multidrug resistance (MDR) profile by adriamycin-selected variants of a human squamous lung carcinoma, an ovarian carcinoma, a cervical carcinoma and by a colchicine-selected variant of a Chinese hamster ovarian carcinoma resulted in alterations to their radiosensitivity. However, the degree of change in the radiosensitivity of the MDR cell lines could not be predicted from their level of resistance to adriamycin. Clonal populations derived from DLKP-A, an adriamycin-selected MDR variant of the human lung carcinoma cell line DLKP, exhibited individual radiosensitivity profiles, which did not correlate with their chemoresistance. Exposure of DLKP to consecutive increasing doses of radiation did not confer cross-resistance to chemotherapeutic drugs.Abbreviations LD₅₀ Radiation dose which kills 50% of the cell population - IC₅₀ Chemotherapeutic drug concentration which kills 50% of the cell population - MDR Multidrug resistance - Gy Gray - EMS Ethyl methanesulfonate     

Seasonal changes in numbers and the location of a particular bacterial strain of Alteromonas sp. in seagrass sediments

Many variables must be considered in seeking to describe differences in population sizes for native aquatic bacterial populations. In this study of seagrass- and nearby plant-free sediments, seasonal effects on total bacterial counts were found to be highly significant, outweighing the significance of factors such as geographic variability, but on populations of a chosen Alteromonas sp., they were not significant at the 5% level. Summer counts for both populations were higher than those for winter; this result is likely to reflect the higher productivity of the host Zostera capricomi in summer months, resulting in the exudation of increased amounts of organic nutrients. The Alteromonas sp. occurred in greatest abundance (1.8% of the total population) at the seagrass sediment site from which it was originally isolated and formed up to 1.5% of the population in adjacent plant-free sediments. In fluorescent microscopy studies with labeled antibodies, the Alteromonas sp. was found to be ubiquitous in seagrass and plant-free sediments but was found closely associated in much higher numbers with seagrass root-rhizome tissue, suggesting a possible nutritional relationship between plant and bacterium. In associated trials of sediment preservation techniques, bacterial counts of replicate sediments preserved with glutaraldehyde (3% v/v) were higher than those obtained using Lugol's iodine or freezing. Correspondence to: P.W. Glazebrook     

Insertion of leader peptidase into the thylakoid membrane during synthesis in a chloroplast translation system

The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum. Lep could thus provide insight into the functional homologies between the different membrane systems. Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E. coli cytoplasmic membranes, following the positive-inside rule. Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes. These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.     

The impact of anchored phylogenomics and taxon sampling on phylogenetic inference in narrow‐mouthed frogs (Anura,Microhylidae)

Despite considerable progress in unravelling the phylogenetic relationships of microhylid frogs, relationships among subfamilies remain largely unstable and many genera are not demonstrably monophyletic. Here, we used five alternative combinations of DNA sequence data (ranging from seven loci for 48 taxa to up to 73 loci for as many as 142 taxa) generated using the anchored phylogenomics sequencing method (66 loci, derived from conserved genome regions, for 48 taxa) and Sanger sequencing (seven loci for up to 142 taxa) to tackle this problem. We assess the effects of character sampling, taxon sampling, analytical methods and assumptions in phylogenetic inference of microhylid frogs. The phylogeny of microhylids shows high susceptibility to different analytical methods and datasets used for the analyses. Clades inferred from maximum‐likelihood are generally more stable across datasets than those inferred from parsimony. Parsimony trees inferred within a tree‐alignment framework are generally better resolved and better supported than those inferred within a similarity‐alignment framework, even under the same cost matrix (equally weighted) and same treatment of gaps (as a fifth nucleotide state). We discuss potential causes for these differences in resolution and clade stability among discovery operations. We also highlight the problem that commonly used algorithms for model‐based analyses do not explicitly model insertion and deletion events (i.e. gaps are treated as missing data). Our results corroborate the monophyly of Microhylidae and most currently recognized subfamilies but fail to provide support for relationships among subfamilies. Several taxonomic updates are provided, including naming of two new subfamilies, both monotypic.     

Phylogenetic patterns of trait and trait plasticity evolution: Insights from amphibian embryos

Environmental variation favors the evolution of phenotypic plasticity. For many species, we understand the costs and benefits of different phenotypes, but we lack a broad understanding of how plastic traits evolve across large clades. Using identical experiments conducted across North America, we examined prey responses to predator cues. We quantified five life‐history traits and the magnitude of their plasticity for 23 amphibian species/populations (spanning three families and five genera) when exposed to no cues, crushed‐egg cues, and predatory crayfish cues. Embryonic responses varied considerably among species and phylogenetic signal was common among the traits, whereas phylogenetic signal was rare for trait plasticities. Among trait‐evolution models, the Ornstein–Uhlenbeck (OU) model provided the best fit or was essentially tied with Brownian motion. Using the best fitting model, evolutionary rates for plasticities were higher than traits for three life‐history traits and lower for two. These data suggest that the evolution of life‐history traits in amphibian embryos is more constrained by a species’ position in the phylogeny than is the evolution of life history plasticities. The fact that an OU model of trait evolution was often a good fit to patterns of trait variation may indicate adaptive optima for traits and their plasticities.     

The Structure of Treponema pallidum Tp0751 (Pallilysin) Reveals a Non-canonical Lipocalin Fold That Mediates Adhesion to Extracellular Matrix Components and Interactions with Host Cells

Syphilis is a chronic disease caused by the bacterium Treponema pallidum subsp. pallidum. Treponema pallidum disseminates widely throughout the host and extravasates from the vasculature, a process that is at least partially dependent upon the ability of T. pallidum to interact with host extracellular matrix (ECM) components. Defining the molecular basis for the interaction between T. pallidum and the host is complicated by the intractability of T. pallidum to in vitro culturing and genetic manipulation. Correspondingly, few T. pallidum proteins have been identified that interact directly with host components. Of these, Tp0751 (also known as pallilysin) displays a propensity to interact with the ECM, although the underlying mechanism of these interactions remains unknown. Towards establishing the molecular mechanism of Tp0751-host ECM attachment, we first determined the crystal structure of Tp0751 to a resolution of 2.15 Å using selenomethionine phasing. Structural analysis revealed an eight-stranded beta-barrel with a profile of short conserved regions consistent with a non-canonical lipocalin fold. Using a library of native and scrambled peptides representing the full Tp0751 sequence, we next identified a subset of peptides that showed statistically significant and dose-dependent interactions with the ECM components fibrinogen, fibronectin, collagen I, and collagen IV. Intriguingly, each ECM-interacting peptide mapped to the lipocalin domain. To assess the potential of these ECM-coordinating peptides to inhibit adhesion of bacteria to host cells, we engineered an adherence-deficient strain of the spirochete Borrelia burgdorferi to heterologously express Tp0751. This engineered strain displayed Tp0751 on its surface and exhibited a Tp0751-dependent gain-of-function in adhering to human umbilical vein endothelial cells that was inhibited in the presence of one of the ECM-interacting peptides (p10). Overall, these data provide the first structural insight into the mechanisms of Tp0751-host interactions, which are dependent on the protein’s lipocalin fold.