Identification of Culturable Oligotrophic Bacteria within Naturally Occurring Bacterioplankton Communities of the Ligurian Sea by 16S rRNA Sequencing and Probing

Abstract Typical marine bacteria (i.e., obligately oligotrophic) that were numerically dominant members of naturally occurring marine communities were identified by cloning and sequencing the amplified 16S rRNA genes obtained from dilution cultures of the original samples. The data reported here refer to two different habitats of a marine pelagic environment (28 miles offshore, in the northwestern Mediterranean Sea). The samples were taken from the water column at two representative layers, i.e., the 30-m depth, corresponding to the chlorophyll maximum layer, and the 1800-m depth, representative of a deep, oligotrophic environment. Three major lineages were found in the 16S rDNA clone libraries prepared from the two samples, two of which could be assigned to the Vibrio and the Rhodobacter groups. The third lineage was a distant relative of the genus Flavobacterium, but it was not closely related to any marine isolate. Six oligonucleotide probes, either complementary to the conserved sequence domains or selectively hybridizing to the clone sequences, were designed for use as hybridization group-specific and strain-specific probes. A single-mismatch discrimination between certain probes and nontarget sequences was demonstrated by detecting the probes' specificity at different hybridization and washing conditions. The screening of the clone libraries with the obtained probes revealed that neither the 30-m sample higher dilution nor the 1800-m one were pure cultures. While some representatives of the Vibrio group were found in both the surface and the deep sample, the members of the Flavobacterium and Rhodobacter lineages were detected only in the deep and the euphotic layers, respectively. We suggest an approach for analyzing autochthonous marine bacteria able to grow in unamended seawater. Received: 19 May 1998; Accepted: 29 October 1998     

Potent inhibitory effects of the 5'-triphosphates of (E)-5-(2-bromovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on DNA polymerase gamma

(E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin. The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP.     

The assessment game in sand fiddler crab contests for breeding burrows

The single enlarged claw of male sand fiddler crabs, Uca pugilator, is used in contests for control of breeding burrows. The larger of the two contestants has the larger claw and usually wins. Males use one or more of 10 agonistic elements that vary in intensity from a no-contact extension of the claw to the flip of an opponent. We used the sequence of elements employed and the duration of unstaged, naturally occurring contests in a South Carolina salt marsh to evaluate three models of extended contests: (1) energetic war of attrition, (2) cumulative assessment and (3) sequential assessment. Contests usually began with elements of low action intensity and often proceeded to elements of high intensity. Elements of higher intensity were correlated with both contest duration and the number of contest elements. Contest duration increased as opponents became more evenly matched in size, a result consistent with both cumulative and sequential assessment models. Variation in duration increased as the relative sizes of opponents increased, also in accordance with sequential assessment. The absolute size of the smaller contestant had no effect on contest duration, in contrast to predictions based on cumulative assessment or energetic war of attrition models. Contestants that lost a fight were more likely to engage immediately in another fight without loss of contest intensity, if their previous fight had been long and intense. This result is inconsistent with contests of endurance, such as the energetic war of attrition or the cumulative assessment game, but it is consistent with the ritualized display of strength and fighting skill. Thus, sequential assessment appears to best explain ritualized fiddler crab contests. Cumulative assessment, however, may be the appropriate model for extended, nonritualized, all-out fights. Cumulative assessment may also explain the tenure of individuals on breeding grounds where multiple engagements are likely to test endurance and tolerance to damage over a period of days. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function; tests for receptor-stimulated phosphatidylinositol turnover and for modulation of agonist binding by guanylylimidodiphosphate showed functional coupling to be present several days prior to the onset of synapse formation. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.     

A numerical taxonomic study of Flavobacterium-Cytophaga strains from dairy sources

Phenotypic data on 203 Gram-negative non-fermentative bacteria of the Flavobacterium-Cytophaga group isolated from milk and butter were analyzed by numerical taxonomic techniques. Twenty reference strains including species of Flavobacterium, Cytophaga and strains of Pseudomonas paucimobilis were included in the study. Using the matching coefficient of Sokal & Michener with antibiotic susceptibility data included, 139 isolates were recovered in nine clusters. Six of these clusters were linked at or above the 85% S level while three were linked at or above the 79% S level. The largest cluster, representing 46.3% of the isolates, could be equated with Flavobacterium sp. Group IIb. Other clusters could be equated with Flavobacterium sp. L 16/1 (22.7% of isolates), F. balustinum (10.8% of isolates), F. breve (4.4%), F. multivorum (3.5%) and Cytophaga johnsonae (1.5%). The cluster resembling Flavobacterium sp. L 16/1 and a smaller unclassified cluster, were exceptional in being susceptible to the antibiotics cephalothin and penicillin G.     

Regulation of voltage-gated Ca channels by lipids

Great skepticism has surrounded the question of whether modulation of voltage-gated Ca²⁺ channels (VGCCs) by the polyunsaturated free fatty acid arachidonic acid (AA) has any physiological basis. Here we synthesize findings from studies of both native and recombinant channels where micromolar concentrations of AA consistently inhibit both native and recombinant activity by stabilizing VGCCs in one or more closed states. Structural requirements for these inhibitory actions include a chain length of at least 18 carbons and multiple double bonds located near the fatty acid's carboxy terminus. Acting at a second site, AA increases the rate of VGCC activation kinetics, and in Ca_V2.2 channels, increases current amplitude. We present evidence that phosphatidylinositol 4,5-bisphosphate (PIP₂), a palmitoylated accessory subunit (β_2a) of VGCCs and AA appear to have overlapping sites of action giving rise to complex channel behavior. Their actions converge in a physiologically relevant manner during muscarinic modulation of VGCCs. We speculate that M₁ muscarinic receptors may stimulate multiple lipases to break down the PIP₂ associated with VGCCs and leave PIP₂'s freed fatty acid tails bound to the channels to confer modulation. This unexpectedly simple scheme gives rise to unanticipated predictions and redirects thinking about lipid regulation of VGCCs.