Hopanoids in marine cyanobacteria: probing their phylogenetic distribution and biological role.

Cyanobacteria are key players in the global carbon and nitrogen cycles and are thought to have been responsible for the initial rise of atmospheric oxygen during the Neoarchean. There is evidence that a class of membrane lipids known as hopanoids serve as biomarkers for bacteria, including many cyanobacteria, in the environment and in the geologic record. However, the taxonomic distributions and physiological roles of hopanoids in marine cyanobacteria remain unclear. We examined the distribution of bacteriohopanepolyols (BHPs) in a collection of marine cyanobacterial enrichment and pure cultures and investigated the relationship between the cellular abundance of BHPs and nitrogen limitation in Crocosphaera watsonii, a globally significant nitrogen-fixing cyanobacterium. In pure culture, BHPs were only detected in species capable of nitrogen fixation, implicating hopanoids as potential markers for diazotrophy in the oceans. The enrichment cultures we examined exhibited a higher degree of BHP diversity, demonstrating that there are presently unaccounted for marine bacteria, possibly cyanobacteria, associated with the production of a range of BHP structures. Crocosphaera watsonii exhibited high membrane hopanoid content consistent with the idea that hopanoids have an important effect on the bulk physical properties of the membrane. However, the abundance of BHPs in C. watsonii did not vary considerably when grown under nitrogen-limiting and nitrogen-replete conditions, suggesting that the role of hopanoids in this organism is not directly related to the physiology of nitrogen fixation. Alternatively, we propose that high hopanoid content in C. watsonii may serve to reduce membrane permeability to antimicrobial toxins in the environment.

Phosphonate utilization by the globally important marine diazotroph Trichodesmium.

The factors that control the growth and nitrogen fixation rates of marine diazotrophs such as Trichodesmium have been intensively studied because of the role that these processes have in the global cycling of carbon and nitrogen, and in the sequestration of carbon to the deep sea. Because the phosphate concentrations of many ocean gyres are low, the bioavailability of the larger, chemically heterogeneous pool of dissolved organic phosphorus could markedly influence Trichodesmium physiology. Here we describe the induction, by phosphorus stress, of genes from the Trichodesmium erythraeum IMS101 genome that are predicted to encode proteins associated with the high-affinity transport and hydrolysis of phosphonate compounds by a carbon-phosphorus lyase pathway. We show the importance of these genes through expression analyses with T. erythraeum from the Sargasso Sea. Phosphonates are known to be present in oligotrophic marine systems, but have not previously been considered to be bioavailable to marine diazotrophs. The apparent absence of genes encoding a carbon-phosphorus lyase pathway in the other marine cyanobacterial genomes suggests that, relative to other phytoplankton, Trichodesmium is uniquely adapted for scavenging phosphorus from organic sources. This adaptation may help to explain the prevalence of Trichodesmium in low phosphate, oligotrophic systems.

The genome of a motile marine Synechococcus.

Marine unicellular cyanobacteria are responsible for an estimated 20-40% of chlorophyll biomass and carbon fixation in the oceans. Here we have sequenced and analysed the 2.4-megabase genome of Synechococcus sp. strain WH8102, revealing some of the ways that these organisms have adapted to their largely oligotrophic environment. WH8102 uses organic nitrogen and phosphorus sources and more sodium-dependent transporters than a model freshwater cyanobacterium. Furthermore, it seems to have adopted strategies for conserving limited iron stores by using nickel and cobalt in some enzymes, has reduced its regulatory machinery (consistent with the fact that the open ocean constitutes a far more constant and buffered environment than fresh water), and has evolved a unique type of swimming motility. The genome of WH8102 seems to have been greatly influenced by horizontal gene transfer, partially through phages. The genetic material contributed by horizontal gene transfer includes genes involved in the modification of the cell surface and in swimming motility. On the basis of its genome, WH8102 is more of a generalist than two related marine cyanobacteria.

Characterization of Trichodesmium spp. by genetic techniques.

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus TRICHODESMIUM: The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of KATAGNYMENE: Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.

Iron stress in open-ocean cyanobacteria (Synechococcus, Trichodesmium, and Crocosphaera spp.): identification of the IdiA protein.

Cyanobacteria are prominent constituents of the marine biosphere that account for a significant percentage of oceanic primary productivity. In an effort to resolve how open-ocean cyanobacteria persist in regions where the Fe concentration is thought to be limiting their productivity, we performed a number of Fe stress experiments on axenic cultures of marine Synechococcus spp., Crocosphaera sp., and Trichodesmium sp. Through this work, we determined that all of these marine cyanobacteria mount adaptive responses to Fe stress, which resulted in the induction and/or repression of several proteins. We have identified one of the Fe stress-induced proteins as an IdiA homologue. Genomic observations and laboratory data presented herein from open-ocean Synechococcus spp. are consistent with IdiA having a role in cellular Fe scavenging. Our data indicate that IdiA may make an excellent marker for Fe stress in open-ocean cyanobacterial field populations. By determining how these microorganisms respond to Fe stress, we will gain insight into how and when this important trace element can limit their growth in situ. This knowledge will greatly increase our understanding of how marine Fe cycling impacts oceanic processes, such as carbon and nitrogen fixation.

Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean.

Fixed nitrogen (N) often limits the growth of organisms in terrestrial and aquatic biomes, and N availability has been important in controlling the CO2 balance of modern and ancient oceans. The fixation of atmospheric dinitrogen gas (N2) to ammonia is catalysed by nitrogenase and provides a fixed N for N-limited environments. The filamentous cyanobacterium Trichodesmium has been assumed to be the predominant oceanic N2-fixing microorganism since the discovery of N2 fixation in Trichodesmium in 1961 (ref. 6). Attention has recently focused on oceanic N2 fixation because nitrogen availability is generally limiting in many oceans, and attempts to constrain the global atmosphere-ocean fluxes of CO2 are based on basin-scale N balances. Biogeochemical studies and models have suggested that total N2-fixation rates may be substantially greater than previously believed but cannot be reconciled with observed Trichodesmium abundances. It is curious that there are so few known N2-fixing microorganisms in oligotrophic oceans when it is clearly ecologically advantageous. Here we show that there are unicellular cyanobacteria in the open ocean that are expressing nitrogenase, and are abundant enough to potentially have a significant role in N dynamics.

Refuting patients' obligations to clinical training: a critical analysis of the arguments for an obligation of patients to participate in the clinical education of medical students.

CONTEXT: The clinical teaching of medical students is essential to the continuation of medicine, but it has a major impact on the patient's health care and autonomy. Some people believe that there is a moral obligation for patients to participate in this training. Such an obligation, real or perceived, may endanger patients' autonomy. OBJECTIVES: The author makes a critical analysis of the main arguments he encounters supporting such an obligation. These arguments are: (1) the furthering of medical education; (2) compensation when uninsured or unable to pay; (3) an equitable return for the care received in a teaching hospital, and (4) fulfilment of a student's need for (and some say right to) clinical training. METHODS: Related literature is reviewed in search of evidence and/or support for such arguments. CONCLUSIONS: The review reveals that these arguments either cannot be verified or do not necessarily place any obligations on the patient. It is argued that, while a medical student may have a right to clinical education, the obligation to fulfil this right rests with the medical university and not on the patients of its teaching hospitals. SOLUTIONS: Several proposals are made about how to satisfy this need without infringing on the patient's right to refuse participation, explaining the patient's rights and role in clinical teaching, and the use of standardized patients where necessary.

Characterization of defects in adult germline development and oogenesis of sterile and rescued female hybrids in crosses between Drosophila simulans and Drosophila melanogaster.

Crosses between Drosophila melanogaster and D. simulans normally result in progeny that are either inviable or sterile. Recent discovery of strains that rescue these inviability and sterility phenotypes has made it possible to study the developmental basis of reproductive isolation between these two species in greater detail. By producing both rescued and unrescued hybrids and examining the protein product staining patterns of genes known to be involved in early germline development and gametogenesis, we have found that in crosses between D. simulans and D. melanogaster, hybrid female sterility results from the improper control of primordial germline proliferation, germline stem cell maintenance, and cystoblast formation and differentiation during early oogenesis. Rescued hybrid females are fertile, yet they generally have lower amounts of adult germline from the outset and show a premature degeneration of adult germline cells with age. In addition, older rescued hybrid females also exhibit mutant egg phenotypes associated with defects in dorso-ventral patterning which may result from the improper partitioning of cytoplasmic factors during early oogenesis that could stem from the early defect. Although a variety of germline and oogenic defects are described for the hybrid females, all of them can potentially result from the same underlying primary defect. Hybrid males from these same crosses, on the other hand, have no detectable germline in adult reproductive tissues, even when hybrid sterility rescue strains are used, indicating that male sterility and female sterility stem from distinctly different developmental defects.

Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma.

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. In the studies reported here, we have examined the role of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway. We asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. We find that Tra(F) is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by Tra(F) can be blocked by the male form of the Dsx protein (Dsx(M)). Expression of the female form of dsx, Dsx(F), in XY animals also induced germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, our findings indicate that the somatic signaling pathway is redundant at the level tra and dsx. Finally, our studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination.

Analysis of the doublesex female protein in Drosophila melanogaster: role on sexual differentiation and behavior and dependence on intersex.

doublesex (dsx) is unusual among the known sex-determination genes of Drosophila melanogaster in that functional homologs are found in distantly related species. In flies, dsx occupies a position near the bottom of the sex determination hierarchy. It is expressed in male- and female-specific forms and these proteins function as sex-specific transcription factors. In the studies reported here, we have ectopically expressed the female Dsx protein (Dsx(F)) from a constitutive promoter and examined its regulatory activities independent of other upstream factors involved in female sex determination. We show that it functions as a positive regulator of female differentiation and a negative regulator of male differentiation. As predicted by the DNA-binding properties of the Dsx protein, Dsx(F) and Dsx(M) compete with each other for the regulation of target genes. In addition to directing sex-specific differentiation, Dsx(F) plays an important role in sexual behavior. Wild-type males ectopically expressing Dsx(F) are actively courted by other males. This acquisition of feminine sex appeal is likely due to the induction of female pheromones by Dsx(F). More extreme behavioral abnormalities are observed when Dsx(F) is ectopically expressed in dsx(-) XY animals; these animals are not only courted by, but also copulate with, wild-type males. Finally, we provide evidence that intersex is required for the feminizing activities of Dsx(F) and that it is not regulated by the sex-specific splicing cascade.

Rapid diversification of marine picophytoplankton with dissimilar light-harvesting structures inferred from sequences of Prochlorococcus and Synechococcus (Cyanobacteria).

Cultured isolates of the unicellular planktonic cyanobacteria Prochlorococcus and marine Synechococcus belong to a single marine picophytoplankton clade. Within this clade, two deeply branching lineages of Prochlorococcus, two lineages of marine A Synechococcus and one lineage of marine B Synechococcus exhibit closely spaced divergence points with low bootstrap support. This pattern is consistent with a near-simultaneous diversification of marine lineages with divinyl chlorophyll b and phycobilisomes as photosynthetic antennae. Inferences from 16S ribosomal RNA sequences including data for 18 marine picophytoplankton clade members were congruent with results of psbB and petB and D sequence analyses focusing on five strains of Prochlorococcus and one strain of marine A Synechococcus. Third codon position and intergenic region nucleotide frequencies vary widely among members of the marine picophytoplankton group, suggesting that substitution biases differ among the lineages. Nonetheless, standard phylogenetic methods and newer algorithms insensitive to such biases did not recover different branching patterns within the group, and failed to cluster Prochlorococcus with chloroplasts or other chlorophyll b-containing prokaryotes. Prochlorococcus isolated from surface waters of stratified, oligotrophic ocean provinces predominate in a lineage exhibiting low G + C nucleotide frequencies at highly variable positions.

Selection and maintenance of sexual identity in the Drosophila germline.

Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transformer 2 gene but, surprisingly, does not appear to require the sex-specific genes, transformer and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-lethal gene.

Combination therapy with zidovudine prevents selection of human immunodeficiency virus type 1 variants expressing high-level resistance to L-697,661, a nonnucleoside reverse transcriptase inhibitor.

L-697,661 is a human immunodeficiency virus type 1 (HIV-1)-specific nonnucleoside reverse transcriptase (RT) inhibitor. Its tolerability and activity in combination with zidovudine were evaluated in a 48-week double-blind study. One hundred nineteen zidovudine-naive HIV-1-infected patients with CD4 cell counts of 200-500/mm3 received either combination therapy, L-697,661 alone, or zidovudine alone. Activity was assessed by CD4 cell count changes. Selection for L-697,661-resistant virus was monitored by susceptibility testing of RT expressed by circulating viral RNA. Therapy was generally well tolerated. All groups receiving zidovudine exhibited transient increases in CD4 cell counts, while the L-697,661 monotherapy group showed a significant decline and yielded RT > 100-fold resistant to L-697,661 and associated with substitutions at RT residue 181. The RT from patients receiving combination therapy was maximally 15-fold less susceptible to L-697,661. Hence, cotreatment with zidovudine prevents selection of HIV-1 variants that are highly resistant to L-697,661 in patients naive to both compounds.

Turnover of circulating virion RNA and of cell-associated viral DNA reflects active viral replication in human immunodeficiency virus type 1-infected individuals.

A quantitative assessment of human immunodeficiency virus type 1 turnover in patient cell-free virion and infected-cell compartments under the dynamic conditions imposed by an effective antiviral therapy was performed. The turnover was rapid, and following a temporal lag, the extent of viral population replacement was eventually similar in both compartments. Each compartment therefore reflects considerable active virus replication.

Resistance to co-occurring phages enables marine synechococcus communities to coexist with cyanophages abundant in seawater.

Recent reports documenting very high viral abundances in seawater have led to increased interest in the role of viruses in aquatic environments and a resurgence of the hypothesis that viruses are significant agents of bacterial mortality. Synechococcus spp., small unicellular cyanobacteria that are important primary producers at the base of the marine food web, were used to assess this hypothesis. We isolated a diverse group of Synechococcus phages that at times reach titers of between 10 and 10 cyanophages per ml in both inshore and offshore waters. However, despite their diversity and abundance, we present evidence in support of the hypothesis that lytic phages have a negligible effect in regulating the densities of marine Synechococcus populations. Our results indicate that these bacterial communities are dominated by cells resistant to their co-occurring phages and that these viruses are maintained by scavenging on the relatively rare sensitive cells in these communities.

Comprehensive mutant enzyme and viral variant assessment of human immunodeficiency virus type 1 reverse transcriptase resistance to nonnucleoside inhibitors.

The nonnucleoside reverse transcriptase (RT) inhibitors comprise a class of structurally diverse compounds that are functionally related and specific for the human immunodeficiency virus type 1 RT. Viral variants resistant to these compounds arise readily in cell culture and in treated, infected human. Therefore, the eventual clinical usefulness of the nonnucleoside inhibitors will rely on a thorough understanding of the genetic and biochemical bases for resistance. A study was performed to assess the effects of substitutions at each RT amino acid residue that influences the enzyme's susceptibility to the various nonnucleoside compounds. Single substitutions were introduced into both purified enzyme and virus. The resulting patterns of resistance were markedly distinct for each of the tested inhibitors. For instance, a > 50-fold loss of enzyme susceptibility to BI-RG-587 was engendered by any of four individual substitutions, while the same level of relative resistance to the pyridinone derivatives was mediated only by substitution at residue 181. Similarly, substitution at residue 181. Similarly, substitution at residue 106 had a noted effect on virus resistance to BI-RG-587 but not to the pyridinones. The opposite effect was mediated by a substitution at residue 179. Such knowledge of nonucleoside inhibitor resistance profiles may help in understanding the basis for resistant virus selection during clinical studies of these compounds.

Development of the formalin-inactivated hepatitis A vaccine, VAQTA from the live attenuated virus strain CR326F.

The development of the formalin-inactivated hepatitis A vaccine, VAQTA, culminates nearly two decades of the basic science studies of VAQTA in hepatitis A virology at the MRL. The master seed virus for production of VAQTA is derived from the F'(P18) variant of the strain CR326F which has been studied in human clinical trials and shown to the highly attenuated. The antigen is highly purified to make possible the consistency and thoroughness of its inactivation by formalin. Phase I clinical studies of VAQTA were initiated in 1989 and have progressed since that time to the recent Phase III clinical trials which demonstrated efficacy of a single dose of the vaccine in preventing clinical hepatitis A disease in pediatric populations in Monroe, NY.

Evaluation of a microcarrier process for large-scale cultivation of attenuated hepatitis A.

Microcarrier culture was investigated for the propagation of attenuated hepatitis A vaccine in the anchorage-dependent human fibroblast cell line, MRC-5. Cells were cultivated at 37 degrees C for one to two weeks, while virus accumulation was performed at 32 degrees C over 21 to 28 days. The major development focus for the microcarrier process was the difference between the cell and virus growth phases. Virus antigen yields, growth kinetics, and cell layer/bead morphology were each examined and compared for both the microcarrier and stationary T-flask cultures. Overall, cell densities of 4-5 x 10(6) cells/ml at 5-10 milligrams beads were readily attained and could be maintained in the absence of infection at either 37 degrees C or 32 degrees C. Upon virus inoculation, however, substantial cell density decreases were observed as well as 2.5 to 10-fold lower per cell and per unit surface area antigen yields as compared to stationary cultures. The advantages as well as the problems presented by the microcarrier approach will be discussed.

Phylogenetic characterization and in situ localization of the bacterial symbiont of shipworms (Teredinidae: Bivalvia) by using 16S rRNA sequence analysis and oligodeoxynucleotide probe hybridization.

It has been proposed that a bacterium isolated from the gills of shipworms (teredinid mollusks) is, by virtue of its ability both to degrade cellulose and to fix dinitrogen, the symbiont that enables these mollusks to utilize wood as their principal food source. The phylogenetic affiliation of four of these bacteria isolated from wood-boring bivalve mollusks was determined by 16S rRNA sequence analysis by using the reverse transcriptase method with six oligodeoxynucleotide primers. The four bacterial strains tested had indistinguishable 16S rRNA sequences, supporting the previous conclusion, based on phenotypic characterization, that these isolates represent a single species. Evolutionary distance matrix analysis of the RNA sequence indicated that the bacterial symbiont falls within the gamma-3 subdivision of the Proteobacteria and is distinct from other known bacterial genera. In situ localization of the bacterial symbiont in tissue sections of the shipworm Lyrodus pedicellatus was determined by using a 16S rRNA-directed oligodeoxynucleotide hybridization probe specific for the bacterium isolated from shipworm gill tissue. Fluorescence microscopy showed that the specific probe bound to L. pedicellatus tissue at sites coincident with the location of symbiont cells and that it did not bind to other host tissues. This technique provided direct visual evidence that the cellulolytic, nitrogen-fixing bacterial isolates were the symbionts observed within the gill of L. pedicellatus.