Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.     

Chemical signals mediating interactions betweenGaleruca tanaceti L. (Coleoptera, Chrysomelidae) and its egg parasitoidOomyzus galerucivorus (Hedqvits) (Hymenoptera, Eulophidae)

Chemical signals mediating interactions betweenGaleruca tanaceti and its egg parasitoidOomyzus galerucivorus (Hymenoptera, Eulophidae) were studied. Neither odor of gravid females ofG. tanaceti nor volatiles of their feces were attractive to the parasitoid. However, the presence of the beetles’ feces on a substrate arrested the parasitoid and elicited frequent antennal drumming. Thus, feces may contain a kairomone important for host finding. Odors of damaged and undamaged host plants had no effect on the parasitoids.O. galerucivorus did not detect its host eggs at close range but encountered them by chance. Neither the structure nor the dark color of the egg surface play a key role in host recognition, but chemicals of the extrachorion which could be isolated by dichloromethane. Fractionation of the dichloromethane extract by TLC revealed a single active fraction which induced host recognition behavior. Since the eggs ofG. tanaceti contain anthraquinones and anthrones which are active as feeding deterrents against predators, we hypothesized that reproductive success ofO. galerucivorus is due to sequestration of these protective compounds. However, GC-MS analyses revealed that there was no transfer of them from the host egg into the adult parasitoid.     

Das Vorkommen von Bacteriochlorophyll aP und aGg in Stämmen aller Arten der Rhodospirillaceae

Summary The bacteriochlorophylls a of 60 strains belonging to 13 different species of the purple nonsulfur bacteria (Rhodospirillaceae) were studied with respect to the nature of the esterifying alcohol. The new bacteriochlorophyll a_Gg containing all-trans-geranylgeraniol is the main bacteriochlorophyll in all strains of Rhodospirillum rubrum. Rhodospirillum photometricum contains the new and the classical bacteriochlorophyll a_P (phytol as esterifying alcohol) in nearly equal amounts. The strains of all other species contain the classical bacteriochlorophyll a_P.     

Phytochrome control of its own accumulation and leaf expansion in tentoxin- and norflurazon-treated mung bean seedlings

Primary leaves of 4-day-old, dark-grown mung bean [ Vigna radiata (L.) Wilczek cv. Berken] seedlings were exposed to 24 h of white light (200 μmol m⁻² s⁻¹) which was terminated by a 15 min, phytochrome-saturating red or far-red light exposure. Phytochrome content (in vivo and in vitro) and leaf area were monitored during the subsequent dark period. Red light treatments resulted in lower phytochrome content and greater leaf expansion than did far-red treatments. Phytochrome accumulation and leaf expansion were less in norflurazon- (no carotenoids and very low Chl) than in tentoxin- (very low Chl) treated leaves. After 3 days of darkness, leaf expansion was about 25% greater and phytochrome content was about 50% less in red- than in far-red-treated leaves of all treatments. These effects generally took longer to develop in norflurazon- than in tentoxin-treated tissues. Norflurazon-treated tissues exposed to long white light periods apparently do not as accurately reflect phytochrome-controlled photomorphogenic events of green tissues as do tentoxin-treated tissues of mung bean seedlings.     

Independent regulation of serologically distinct idiotypes in F1 hybrid mice

We have investigated the regulation of expression of two distinct intrastrain cross-reactive idiotypes, CRI_A and CRI_C, characteristic of anti-p-azophenylarsonate (anti-Ar) antibodies of the A/J and BALB/c strains, respectively, in (BALB/c x A/J)F₁ (CAF₁) mice. Such hybrid mice were found to synthesize antibodies with each idiotype when immunized against the Ar hapten group, although the expression of each was significantly reduced as compared with the parental strain. CAF₁ mice were pretreated with idiotypic-specific antibody reagents and subsequently hyperimmunized against the Ar hapten. Analysis of the idiotypes present in immune sera showed that suppression of either CRI did not concomitantly suppress the expression of the other. Alteration of the expression of one idiotype was not, however, without influence on the other; the expression of CRI_C was markedly enhanced in mice suppressed for CRI_A.Abbreviations used in this paper anti-Id(A/J) idiotypic-specific antibodies against A/J serum Ar-specific antibodies - anti-Id(BALB/c) idiotypic-specific antibodies against BALB/c serum Ar-specific antibodies - Ar p-azophenylarsonate - BGG bovine -globulin - BSA bovine serum albumin - CAF₁ F(BALB/c x A/J) - CFA complete Freund's adjuvant - CRIA the major cross-reactive idiotype of A/J Ar-specific antibodies - CRI_C the major cross-reactive idiotype of BALB/c Ar-speck antibodies - CRI_m the minor cross-reactive idiotype of A/J Ar-specific antibodies - DTH delayed-type hypersensitivity - HP hybridoma product(s) - KLH keyhole limpet hemocyanin     

Comparison of stem-cell recovery in autoimmune and normal strains

The capacity of NZB stem cells to proliferate in vivo was evaluated in two systems which required repopulation of peripheral organs. In both types of depletion systems, stem-cell repopulation after cyclophosphamide treatment or adoptive transfer repopulation in lethally irradiated hosts, it was found that NZB stem cells were hyperproliferating. The increase in proliferating cells was most pronounced in the spleens of NZB mice treated with high-dose cyclophosphamide and in lethally irradiated F₁ mice reconstituted with NZB T-cell-depleted bone marrow. Thus, upon a stimulus to repopulate, NZB marrow stem cells will hyperproliferate in peripheral organs resulting in an increase in cell number. The abnormality in the marrow cells can be observed in young NZB mice when their marrow cells are in an environment which requires recovery and division.     

Development of chicken intrafusal muscle fibers

The first sign of developing intrafusal fibers in chicken leg muscles appeared on embryonic day (E) 13 when sensory axons contacted undifferentiated myotubes. In sections incubated with monoclonal antibodies against myosin heavy chains (MHC) diverse immunostaining was observed within the developing intrafusal fiber bundle. Large primary intrafusal myotubes immunostained moderately to strongly for embryonic and neonatal MHC, but they were unreactive or reacted only weakly with antibodies against slow MHC. Smaller, secondary intrafusal myotubes reacted only weakly to moderately for embryonic and neonatal MHC, but 1–2 days after their formation they reacted strongly for slow and slow-tonic MHC. In contrast to mammals, slow-tonic MHC was also observed in extrafusal fibers. Intrafusal fibers derived from primary myotubes acquired fast MHC and retained at least a moderate level of embryonic MHC. On the other hand, intrafusal fibers developing from secondary myotubes lost the embryonic and neonatal isoforms prior to hatching and became slow. Based on relative amounts of embryonic, neonatal and slow MHC future fast and slow intrafusal fibers could be first identified at E14. At the polar regions of intrafusal fibers positions of nerve endings and acetylcholinesterase activity were seen to match as early as E16. Approximately equal numbers of slow and fast intrafusal fibers formed prenatally; however, in postnatal muscle spindles fast fibers were usually in the majority, suggesting that some fibers transformed from slow to fast.     

Xanthomonas campestris pv. campestris gum Mutants: Effects on Xanthan Biosynthesis and Plant Virulence

Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490–2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.     

Quality control in homograft valve processing: when to screen for microbiological contamination?

Human donor heart valves remain essential for many reconstructive heart procedures. Heart valve donations are a scarce resource which must be used efficiently and safely. Infection transmission remains a potential risk with homograft valve use. Early experience with homograft valves identified high rates of microbial contamination at collection and initiated the practise of immersion in an antibiotic cocktail. Many centres rely on the microbiology screening after exposure to the antibiotic cocktail. We in our centre accept or reject valves on the basis of the microbiology screening at the time of collection prior to immersion in antibiotic solution. We wanted to compare our rate of valve discard and the rate of microbial contamination at implant with other centres. Valves are collected for the Irish Heart Valve Tissue Bank through partnership between the National Centre for Cardiothoracic Surgery and the Irish Blood Transfusion Service. Valves are collected in a surgical theatre setting and processed in dedicated section of the Irish Blood Transfusion Board. Tissues are screening for microbiology at collection and also at implantation. A total of 564 human heart valves and valve conduits were processed through the service during the study period. 167 (29.6%) were discarded during the processing and storage stages. The major reason for this in 117 cases was unsatisfactory microbiology on initial tissue screening. Repeat screening of accepted valves at the time of implantation identified positive cultures in only 0.9%. Optimal use of these limited resources is clearly important. However recipient safety remains paramount. One-fifth of collected valves are discarded at the processing stage due to positive microbiology screening. This is a higher rate of discard then other centres which reject 5.6–10% due to positive microbiology. However our rate of contamination at time of implant is lower then the 3% rate reported elsewhere. We are satisfied that our current discard rate, although significant, reflects rigorous quality control and the optimal balance between valve availability and patient safety.