Reduced Growth of Blastocrithidia culicis and Crithidia oncopelti Freed of Intracellular Symbiotes by Chloramphenicol*

The intracellular symbiotes of Blastocrithidia culicis and Crithidia oncopelti can be eliminated from cultures of the flagellates by a single chloramphenicol (CAP) treatment. Effective dosages were determined to be 0.01–0.08% (w/v) CAP after a treatment for 2 weeks or more for B. culicis and 0.08% (w/v) after 1 month for C. oncopelti in most cases. Ineffective dosages only lowered the numbers of symbiote-bearing flagellates. Growth of both species of flagellates in the presence of CAP was reduced in proportion to the drug concentration. Repeated subcultures at effective dosages yielded symbiote-free flagellates, which maintained a low level of growth rate. After repeated subcultures at ineffective dosages, the growth rate rose and the symbiote-bearing cells, initially very few, increased in number. The lowest effective dosages proved to be marginal, often producing symbiote-free cultures, but occasionally cultures with a few symbiote-bearing cells. After repeated subcultures at these drug concentrations, symbiote-containing cultures grew faster than the symbiote-free cultures. Hence, the symbiotic bacteria benefit the growth of their hosts, perhaps by supplying essential factors that are inadequate even in a rich blood medium.     

Symbiote-Free Hemoflagellates,Blastocrithidia culicis and Crithidia oncopelti: their Liver Factor Requirement and Serologic Identity*

SYNOPSIS. Several aposymbiotic strains of Blastocrithidia culicis and Crithidia oncopelti were cultivated in Trager's chemically defined medium as well as in a blood broth, both supplemented with 0.25% (v/v) liver extract concentrate. For all such strains, the liver extract was found to serve as an essential growth factor in the defined medium and as growth promoting additive in the blood broth. The active molecules were found to be water-soluble, heat stable, dialyzable, and probably nonlipid fractions. Antisera were developed in rabbit against all the available aposymbiotic strains. An almost total cross-reactivity at very high titers was observed in reciprocal agglutination test using strains with and without the bacterial symbiotes. These results indicate that the loss of the symbiotes does not affect the antigenic identity of B. culicis and C. oncopelti.     

Effect of Chloramphenicol on Bacterial Endosymbiotes in a Strain of Amoeba proteus*

SYNOPSIS. The effect of chloramphenicol (CAP) on the bacterial endosymbiotes of a strain of Amoeba proteus was studied by growing the symbiotic amebae in media containing 0.5–1.6 mg/ml CAP for up to 4 weeks. Treatments with CAP caused such ultrastructural changes as expansion of the nuclear zone and deformation of symbiotes. The CAP treatment also damaged the mitochondria, e.g. disappearance of central and protrusion of peripheral cristae. Number of bacteria per ameba decreased to < 10% of control in CAP-containing media, but no viable amebae became completely free of symbiotes. The resuts supported previous studies that amebae were dependent on endosymbiotes.     

Ultrastructural Visualization of Lipids in Trypanosomatids1

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis, T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.     

Omikron,ein essentieller Endosymbiont von Euplotes aediculatus*†

ZUSAMMENFASSUNG. Der Süßwasserciliat Euplotes aediculatus beherbergt in seinem Cytoplasma 900–1000 stäbchenförmige Symbionten, die für seine Vermehrung essentiell zu sein scheinen. Durch Wachstum in Gegenwart von Penicillin kann Euplotes von seinen Symbionten befreit werden, es führt dies jedoch stets auch zu einem Verlust der Teilungsfähigkeit. Eine Reinfektion ist bei etwa 4% der Zellen möglich, die dann nach 4–5 Tagen sich wieder zu vermehren beginnen. Die Endosymbionten wurden sowohl lichtmikroskopisch als auch elektronenmikroskopisch untersucht. In ihrer Feinstruktur und Färbbarkeit gleichen sie, wie kappa und andere von Paramecium her bekannte “killer”-Partikel, gramnegativen Bakterien. Sie unterscheiden sich von diesen Symbionten jedoch dadurch, daß ihre DNA im Cytoplasma nicht homogen verteilt ist, sondern sich in 3–9 schon lichtmikroskopisch erkennbaren Zentren konzentriert. Die in Euplotes vorkommenden Symbionten lassen keine “killer”-Aktivität erkennen. Sie werden omikron-Partikel genannt. SYNOPSIS. The fresh water ciliate Euplotes aediculatus contains in its cytoplasm 900–1000 rod-shaped symbiotes which appear to be essential for division. Growth of Euplotes in the presence of penicillin results in loss of these symbiotes and simultaneously in a loss of the ciliate's ability to divide. Reinfection with the symbiotes can be achieved in 4% of the cells which then resume growth after a lag period of 4–5 days. The endosymbiotes have been studied by light and electron microscopy. In their fine structure and staining reaction they resemble gram-negative bacteria as do kappa and other killer particles of Paramecium. The symbiotes of Euplotes, however, are unusual in that their DNA is not distributed throughout the cytoplasm but is localized in 3–9 areas (nucleoids), which are visible even in the light microscope. No killing activity seems to be associated with the symbiotes. Following the practice of referring to those endosymbiotes by Greek letters they are here designated omikron particles.     

Detritus as a potential food source for protozoans: utilization of fine particulate plant detritus by a heterotrophic flagellate, <Emphasis Type="Italic">Chilomonas paramecium</Emphasis>, and a ciliate, <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>

We investigated the direct utilization of fine particulate detritus (dried and homogenized plant material in the size range of bacteria) as a food source for protozoans using axenic cultures of the cryptomonad, heterotrophic flagellate, Chilomonas paramecium, and the hymenostome ciliate, Tetrahymena pyriformis. When fed media containing only particulate detritus, these species revealed growth rates similar to those reported for field populations. The growth rates of Chilomonas fed exclusively particulate detritus were similar to those obtained on a bacterial diet. Considering the high percentage of detritus particles in the size range of bacteria in lakes, our results imply that direct utilization of detritus by protozoans may form an additional pathway of carbon in aquatic food webs that has generally been overlooked.     

Microbial utilization of the industrial wastewater pollutants 2-ethylhexylthioglycolic acid and <Emphasis Type="Italic">iso</Emphasis>-octylthioglycolic acid by aerobic Gram-negative bacteria

Industrial wastewater from the production of sulfur containing esters and the resulting products of this synthesis, 2-ethylhexylthioglycolic acid (EHTG) and iso-octylthioglycolic acid (IOTG), were deployed in this study to enrich novel bacterial strains, since no wastewater and EHTG or IOTG degrading microorganisms were hitherto described or available. In addition, nothing is known about the biodegradation of these thiochemicals. The effect of this specific wastewater on the growth behaviour of microorganisms was investigated using three well-known Gram-negative bacteria (Escherichia coli, Pseudomonas putida, and Ralstonia eutropha). Concentrations of 5% (v/v) wastewater in complex media completely inhibited growth of these three bacterial strains. Six bacterial strains were successfully isolated, characterized and identified by sequencing their 16S rRNA genes. Two isolates referred to as Achromobacter sp. strain MT-E3 and Pseudomonas sp. strain MT-I1 used EHTG or IOTG, respectively, as well as the wastewater as sole source of carbon and energy for weak growth. More notably, both isolates removed these sulfur containing esters in remarkable amounts from the cultures supernatant. One further isolate was referred to as Klebsiella sp. strain 58 and exhibited an unusual high tolerance against the wastewater’s toxicity without utilizing the contaminative compounds. If cultivated with gluconic acid as additional carbon source, the strain grew even in presence of more than 40% (v/v) wastewater. Three other isolates belonging to the genera Bordetella and Pseudomonas tolerated these organic sulfur compounds but showed no degradation abilities.     

Susceptibility of Rickettsia monacensis and Rickettsia peacockii to Cecropin A, Ceratotoxin A, and Lysozyme

Ticks host obligate intracellular bacteria that range from benign symbiotes to virulent human pathogens. The effects on those bacteria of antimicrobial peptides (AMPs) involved in arthropod innate immunity to microbial infections are largely unknown. We evaluated effects of AMPs and a c-type lysozyme on host cell-free suspensions of the tick symbiotes Rickettsia monacensis and Rickettsia peacockii with stain-based infectivity and viability assays. Cecropin A at a concentration of 8 μM had a lethal effect on both rickettsiae while ceratotoxin A was approximately 20-fold less effective. Toxicity of both AMPs was synergized by lysozyme, an enzyme expressed by ticks. Lactoferrin, a transferrin, had no effect on R. monacensis at up to 110 μM. The rickettsiae were less sensitive to the AMPs than is typical of bacteria that grow extracellularly. Our assays may be useful in the study of AMP activity against other obligate intracellular bacteria.     

Oviposition substrate selection by Florida mosquitoes in response to pathogen‐infected conspecific larvae

The impact of the presence of larval mosquito pathogens with potential for biological control on oviposition choice was evaluated for three mosquito species/pathogen pairs present in Florida. These included Aedes aegypti infected with Edhazardia aedis, Aedes albopictus infected with Vavraia culicis, and Culex quinquefasciatus infected with Culex nigripalpus nucleopolyhedrovirus (CuniNPV). Two‐choice oviposition bioassays were performed on each host and pathogen species with one oviposition cup containing infected larvae and the other cup containing uninfected larvae (control). Both uninfected and E. aedis‐infected female Ae. aegypti laid significantly fewer eggs in oviposition cups containing infected larvae. Uninfected gravid female Ae. albopictus and Cx. quinquefasciatus oviposited equally in cups containing uninfected larvae or containing larvae infected with V. culicis or CuniNPV, respectively. Gravid female Ae. albopictus infected with V. culicis did not display ovarian development and did not lay eggs. The decreased oviposition by gravid Ae. aegypti in containers containing E. aedis‐infected larvae may indicate that the infected larvae produce chemicals deterring oviposition.     

Resuscitation of amebae deprived of essential symbiotes: Micrurgical studies*

SYNOPSIS. The effect of depletion and restoration of obligatory bacterial endosymbiotes on Amoeba proteus strain xD was studied. Removal of the symbiotes by culturing the amebae at 26.5 C resulted in loss of viability of the host cells, indicating that this strain is dependent on its endosymbiotes for survival. Amebae depleted of bacteria could initially be resuscitated by injection of isolated symbiotes, but prolonged deprivation led to irreversible changes. Nuclei of aposymbiotic amebae were viable when transplanted into the cytoplasm of normal cells, but the symbiote-depleted cytoplasm of heat-treated amebae could not be resuscitated by renucleation. No immediate ultrastructural changes were detected in aposymbiotic amebae except for clumping of nucleoli. Thus it appears that the symbiote performs an essential function as a cytoplasmic constituent.     

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase

Aims: 1‐Aminocyclopropane‐1‐carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. Methods and Results: A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat‐resistant polypropylene chimney‐top 96‐well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC‐utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC‐utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Conclusions: Determination of bacterial ACC consumption by the PCR‐plate ninhydrin–ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. Significance and Impact of the Study: The PCR‐plate ninhydrin–ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates.     

Identification of Two Species of Yeast-like Symbiotes in the Brown Planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis>

To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed spacer (ITS)–5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that the two different 18S and ITS–5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and ITS–5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS–5.8S rDNA, showed the high similarity to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes.     

Phylogenetic affiliation of BEV,a bacterial parasite of the leafhopperEuscelidius variegatus,on the basis of 16S rDNA sequences

The phylogenetic relationship of a nonflagellated, Gram-negative, rod-shaped intracellular bacterial parasite (BEV) of the leafhopperEuscelidius variegatus to other bacteria within the classProteobacteria was determined by sequence analysis of 16S rDNAs. The presence of specific signature nucleotides showed this bacterium to be a member of the -3 subdivision of theProteobacteria. Phylogenetic analysis based on maximum parsimony placed BEV within a clade in theEnterobacteriaceae, which includes a number of bacteria that are facultative symbiotes of insects and have a common ancestor withProteus vulgaris. Within this clade, BEV is most closely related to a bacterium identified as the secondary endosymbiote of another homopteran, the pea aphid,Acyrthosiphon pisum.     

Ultrastructure of the Bacterial Symbiotes in the Pharyngeal Diverticulum of Dacus oleae (Gmelin) (Trypetidae; Diptera)

Abstract The ultrastructure of the bacterial symbiotes in the pharyngeal diverticula of adult olive flies [Dacus oleae (Gmelin)] was examined. The diverticulum was an extension of the foregut formed by a row of epithelial cells bounded by an inner layer of cuticle. Towards the hemolymph, the epithelial cells showed an infolding of their basement membrane while adjacent to the cuticular lining, the cells contained a zone of extensive membrane proliferation. The diverticula were packed with bacterial rods which possessed elongate filamentous and short catenulate appendages. The function of these appendages is unknown. They did not resemble fimbriae (pili), flagella or prosthecae described from other bacteria.     

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200?rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent K_m and V_max values of the cutinase for p-nitrophenyl butyrate were 0.72?mM and 336.8?µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.     

Fluorescence microscopy of plastid nucleoids and a survey of nuclease C in higher plants with respect to mode of plastid inheritance

Summary Plastid nucleoids (pt nucleoids) were observed during pollen formation, or in generative cells of mature pollen grains using fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI). Nuclease C activity was surveyed using SDS-PAGE and agarose gel nuclease assay methods. InMirabilis jalapa, pt nucleoids were observed both in pollen mother cells and the monocellular pollen grains after meiosis, followed by the complete disappearance both in the generative and vegetative cells at the bicellular pollen grain stage. This observation is a direct evidence of maternal plastid inheritance. By contrast, in the generative cells of mature pollen grains fromRhododendron kaempferi, Zygocactus truncatus, Oenothera laciniata, andO. speciosa, pt nucleoids were clearly observed. Thus cytological evidence convinces the mode of biparental plastid inheritance. Nuclease C activity was clearly detected both in the stamen and pistil ofM. jalapa. InR. kaempferi low nuclease C activity was detected in both organs, but the activity in the stamen was much less than in the pistil. InZ. truncatus, O. laciniata, andO. speciosa, the activities were difficult to detect in both organs. These results suggest a significant role of nuclease C for the digestion of pt nucleoids in the generative cells.Abbreviations EGTA ethylene-glycol-bis-(2-aminoethyl ether)-N, N, N, N-tetraacetic acid - DAPI 4,6-diamidino-2-phenylindole - Nuclease C Ca²⁺ dependent nuclease - SDS-PAGE SDS-polyacrylamide gel electrophoresis - pt nucleoids plastid nucleoids     

Nutritional Requirements of Blastocrithidia culicis,a Trypanosomatid with an Endosymbiont1

ABSTRACT. The symbiont-containing Blastocrithidia culicis from Aedes vexans, unlike most lower trypanosomatids cultivated in defined media, require only three amino acids: methionine, histidine, and arginine; only three vitamins: thiamin, nicotinamide, and riboflavin; and neither heme nor purine. The bacterium-like endosymbiont presumably provides the trypanosomatid's other essential nutrients.     

Isolation and characterization of symbiotes from the Rocky Mountain wood tick, Dermacentor andersoni

Wolbachia-like symbiotes in the Rocky Mountain wood tick, Dermacentor andersoni, were isolated repeatedly by injection of ovarial tissues into 5-day-old chick embryos. In Giemsa-stained smears of infected embryo tissues, the organisms appeared as blueish or pinkstained coccal bodies indistinguishable from those seen in the ovaries of ticks, where they are located in the luminal epithelium and funicle cells, as well as in oocytes.Electron microscopy revealed that these symbiotes are highly pleomorphic and vary in size from 0.6 to 3.4 μm in diameter. Their fine structure in tissue cells is differentiated into a granular, cortical region, which contains densely stained ribosomes, and a medullary region consisting of a diffuse reticulum partially or completely devoid of granular material or ribosomes. Multiplication is by binary fission. Each organism is delimited by a distinct plasmalemma; a cell wall as in bacterial and rickettsial agents was not observed in organisms from ovarial tissues.Symbiotes cultivated in chick embryos and then injected intracoelomically into adult D. andersoni, developed rapidly and produced massive infestations in hemocytes, hypodermal tissues, salivary glands, and in connective tissues surrounding midgut, Malpighian tubules, and ovary. In hypodermal tissue, organisms with a distinct bilayered cell envelope were occasionally detected. The massive invasion of tissues by injected symbiotes invariably proved fatal for ticks.Results of complement-fixation tests and of fluorescent antibody staining indicated that symbiotes in D. andersoni are closely related to Wolbachia persica, previously isolated from Argas arboreus.     

A second polycaprolactone depolymerase from Fusarium, a lipase distinct from cutinase

Polycaprolactone (PCL), a synthetic polyester with applications in biodegradable plastics, is degraded by a variety of microorganisms, including fungal phytopathogens. These pathogens secrete cutinase, which hydrolyzes cutin, the polyester structural component of plant cuticle, releasing ω-hydroxy fatty acids that induce cutinase synthesis. Our laboratory previously reported that growth of Fusarium solani on PCL requires cutinase, which is active as a PCL depolymerase and induced by the products of its action on PCL. A mutant strain of F. solani in which the cutinase gene is deleted was unable to grow on PCL and did not secrete PCL depolymerase activity in the media tested. It is now shown that this mutant produces a PCL depolymerase in media containing lipase inducers. Wild-type strains also produce this second PCL depolymerase, which is induced by Tween 80 and tributyrin, but not by PCL or cutin. The second depolymerase shows interfacial activation, indicating that it is a lipase. PCL may thus be a substrate but not an inducer of depolymerases that degrade it, and screening microorganisms on medium with PCL as the sole source of carbon and energy may fail to reveal strains with active PCL depolymerases, because of the absence of an inducer. Surprisingly, Tween 80 induces both cutinase and lipase activities in wild-type F. solani. Received: 31 March 1998 / Received revision: 27 July 1998 / Accepted: 8 August 1998     

DNA-Binding Proteins and Structural Characteristics of Chloroplast Nucleoids

A comparative analysis of proteins from chloroplast nucleoids was performed in two higher-plant species (Pisum sativumL. andArabidopsis thalianaL.) and a green alga Chlamydomonas reinhardtiiDang. In the nucleoids of the higher plants and the alga, 26–27 proteins were detected with their mol wts ranging from 10 to more than 94 kD. In all the species tested, the distribution of nucleoid proteins by their mol wts was similar, especially between the predominant proteins with mol wts of 10 to 40 kD. Six DNA-binding proteins (12–18 kD) were detected in nucleoids fromCh. reinhardtiichloroplasts after in vivocovalent cross-linking between chloroplast DNA and proteins. Under an electron miscroscope, some regular structures resembling nucleosome-like particles of bacterial cells were revealed.