Determination of Active Marine Bacterioplankton: a Comparison of Universal 16S rRNA Probes, Autoradiography, and Nucleoid Staining

We compared several currently discussed methods for the assessment of bacterial numbers and activity in marine waters, using samples from a variety of marine environments, from aged offshore seawater to rich harbor water. Samples were simultaneously tested for binding to a fluorescently labeled universal 16S rRNA probe; (sup3)H-labeled amino acid uptake via autoradiography; nucleoid-containing bacterial numbers by modified DAPI (4(prm1),6-diamidino-2-phenylindole) staining; staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), a compound supposed to indicate oxidative cell metabolism; and total bacterial counts (classical DAPI staining), taken as a reference. For the universal-probe counts, we used an image intensifying and processing system coupled to the epifluorescence microscope. All of the above-mentioned methods yielded lower cell counts than DAPI total counts. Universal-probe counts averaged about half of the corresponding DAPI count and were highly correlated to autoradiography counts (r(sup2) = 0.943; n = 7). Nucleoid-containing cell counts could be lower than DAPI counts by as much as 1 order of magnitude but sometimes matched autoradiography or probe counts. CTC counts were 2 orders of magnitude below DAPI counts. Universal 16S rRNA probe counts correlated well with autoradiography results, indicating a population with at least minimal metabolic activity. The greater variability of the nucleoid-containing cell counts calls for further investigation of the processes involved, and CTC counts were well below the range of the other methods tested.     

Bacterial activity along a trophic gradient

Bacterial biomass, secondary production, and extracellular enzymatic activity [-glucosidase and leucine-aminopeptidase, measured as cleavage of artificial fluorogenic substrates 4-methyl umbelliferyl (MVF) -D-glucopyranoside and L-leucine 7-amido-4-methyl coumarin (MCA)] were measured along a trophic gradient in the Northern Adriatic Sea in four ecologically different situations. Bacterial parameters were compared with chlorophyll a and inorganic and organic nutrient concentrations. Bacterial secondary production and extracellular enzymatic activity markedly changed among different seasons and along the trophic gradient. Average bacterial secondary production increased from 0.61 to 2.09 µg Cl^–1 hour^–1 preceding a bloom, to 2.09 µg Cl^–1 hour^–1 during the bloom, decreasing again to 0.81 and 0.83 µg Cl^–1 hour^–1 in the post-bloom and summer periods, respectively (values from 0.5 m depth). Leucine-aminopeptidase activity showed more consistent trends than -glucosidase activity. Average values of leucine-aminopeptidase activity, measured by enzymatic release of MCA, increased from a pre-bloom value of 164.0 to 1,712.0 (nM MCA) hour^–1 released during a bloom, decreasing to 298.5 and 133.7 (nM MCA) hour^–1 released for the post-bloom and summer situation, respectively (values from 0.5 m depth). Average growth rates decreased during the bloom, whereas average extracellular enzymatic activity levels expressed on a cell basis increased by an average factor of 2. Along the trophic gradient, a consistent increase in bacterial secondary production could be observed in all but the summer situation (values from 0.5 m depth). Leucine-aminopeptidase activity also showed positive trends along the gradient, while -glucosidase activity did not exhibit such a clear trend. Bacterial biomass trends were less obvious considering both seasonal changes and the tropic gradient. Highly significant interrelations were detected between bacterial proteolytic activity, secondary production, chlorophyll a content, and nitrate concentrations, especially in the surface horizon. Send offprint requests to: G. J. Hemdl.     

Cross entropy minimization in uninvadable states of complex populations

Selection is often. viewed as a process that maximizes the average fitness of a population. However, there are often constraints even on the phenotypic level which may prevent fitness optimization. Consequently, in evolutionary game theory, models of frequency dependent selection are investigated, which focus on equilibrium states that are characterized by stability (or uninvadability) rather than by optimality. The aim of this article is to show that nevertheless there is a biologically meaningful quantity, namely cross (fitness) entropy, which is optimized during the course of evolution: a dynamical model adapted to evolutionary games is presented which has the property that relative entropy decreases monotonically, if the state of a (complex) population is close to an uninvadable state. This result may be interpreted as if evolution has an order stabilizing effect.     

The relationship between photosynthesis and a mastoparan-induced hypersensitive response in isolated mesophyll cells

The G-protein activator mastoparan (MP) was found to elicit the hypersensitive response (HR) in isolated Asparagus sprengeri mesophyll cells at micromolar concentrations. The HR was characterized by cell death, extracellular alkalinization, and an oxidative burst, indicated by the reduction of molecular O₂ to O₂⁻. To our knowledge, this study was the first to monitor photosynthesis during the HR. MP had rapid and dramatic effects on photosynthetic electron transport and excitation energy transfer as determined by variable chlorophyll a fluorescence measurements. A large increase in nonphotochemical quenching of chlorophyll a fluorescence accompanied the initial stages of the oxidative burst. The minimal level of fluorescence was also quenched, which suggests the origin of this nonphotochemical quenching to be a decrease in the antenna size of photosystem II. In contrast, photochemical quenching of fluorescence decreased dramatically during the latter stages of the oxidative burst, indicating a somewhat slower inhibition of photosystem II electron transport. The net consumption of O₂ and the initial rate of O₂ uptake, elicited by MP, were higher in the light than in the dark. These data indicate that light enhances the oxidative burst and suggest a complex relationship between photosynthesis and the HR.     

Detection, purification and efficacy of warnerin produced by Staphylococcus warneri

Summary Three strains of Staphylococcus warneri (FM10, FM20 and FM30) isolated from meat samples were investigated for their ability to synthesize bacteriocin. All the tested strains produced warnerin, a new peptide bacteriocin; which inhibits the growth of a large number of Gram-positive and Gram-negative bacteria. The inhibitory effect of warnerin produced by the FM20 isolate was high when compared to the other isolates. The results on the effect of carbon sources, nitrogen sources, pH, temperature, incubation time and surfactant (tween 80) inferred that the bacteriocin production was high in medium supplemented with 1% glucose (12,800 AU/ml), 1% urea (6800 AU/ml), and 0.5% Tween 80 (25,600 AU/ml). The higher productivity of bacteriocin was registered during 12 h of incubation in the medium pH 6.5 at 37 °C temperature. Among the various indicator strains tested, Staphylococcus aureus was more sensitive to the bacteriocin activity. Partially purified warnerin exhibited a single band on SDS-PAGE with an apparent molecular weight of 2500 Da. Warnerin, the antibacterial compound was determined as a proteinaceous substance, since it lost its activity when pepsin was added.     

A graph-theoretic approach to comparing and integrating genetic, physical and sequence-based maps

For many species, multiple maps are available, often constructed independently by different research groups using different sets of markers and different source material. Integration of these maps provides a higher density of markers and greater genome coverage than is possible using a single study. In this article, we describe a novel approach to comparing and integrating maps by using abstract graphs. A map is modeled as a directed graph in which nodes represent mapped markers and edges define the order of adjacent markers. Independently constructed graphs representing corresponding maps from different studies are merged on the basis of their common loci. Absence of a path between two nodes indicates that their order is undetermined. A cycle indicates inconsistency among the mapping studies with regard to the order of the loci involved. The integrated graph thus produced represents a complete picture of all of the mapping studies that comprise it, including all of the ambiguities and inconsistencies among them. The objective of this representation is to guide additional research aimed at interpreting these ambiguities and inconsistencies in locus order rather than presenting a "consensus order" that ignores these problems.     

Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells

The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of alpha3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.     

Decreased binding of peptides-MHC class I (pMHC) multimeric complexes to CD8 affects their binding avidity for the TCR but does not significantly impact on pMHC/TCR dissociation rate

The CD8 coreceptor plays a crucial role in both T cell development in the thymus and in the activation of mature T cells in response to Ag-specific stimulation. In this study we used soluble peptides-MHC class I (pMHC) multimeric complexes bearing mutations in the CD8 binding site that impair their binding to the MHC, together with altered peptide ligands, to assess the impact of CD8 on pMHC binding to the TCR. Our data support a model in which CD8 promotes the binding of TCR to pMHC. However, once the pMHC/TCR complex is formed, the TCR dominates the pMHC/TCR dissociation rates. As a consequence of these molecular interactions, under physiologic conditions CD8 plays a key role in complex formation, resulting in the enhancement of CD8 T cell functions whose specificity, however, is determined by the TCR.     

Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis

Fluorescence-labeled soluble major histocompatibility complex class I-peptide "tetramers" constitute a powerful tool to detect and isolate antigen-specific CD8(+) T cells by flow cytometry. Conventional "tetramers" are prepared by refolding of heavy and light chains with a specific peptide, enzymatic biotinylation at an added C-terminal biotinylation sequence, and "tetramerization" by reaction with phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show here that such preparations are heterogeneous and describe a new procedure that allows the preparation of homogeneous tetra- or octameric major histocompatibility complex-peptide complexes. These compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which recognize the Plasmodium berghei circumsporzoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on Lys(259) in the context of H-2K(d). We report that mutation of the CD8 binding site of K(d) greatly impairs the binding of tetrameric but not octameric or multimeric K(d)-PbCS(ABA) complexes to CTLs. This mutation abolishes the ability of the octamer to elicit significant phosphorylation of CD3, intracellular calcium mobilization, and CTL degranulation. Remarkably, however, this octamer efficiently activates CTLs for Fas (CD95)-dependent apoptosis.     

CD3 delta establishes a functional link between the T cell receptor and CD8

T cells expressing T cell receptor (TCR) complexes that lack CD3 delta, either due to deletion of the CD3 delta gene, or by replacement of the connecting peptide of the TCR alpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8 beta or tailless CD8 beta, we examined whether CD3 delta serves to couple TCR.CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K(d). We report that, in thymocytes and hybridomas expressing the T1 TCR.CD3 complex, CD8 alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8 alpha alpha or a CD3 delta(-) variant of the T1 TCR. CD3 delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3 delta. Because CD8 alpha beta is a raft constituent, due to this association a fraction of TCR.CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3 delta couples TCR.CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8(+) T cells.