Isolation and composition of bacteriophage-like particles from kappa of killer Paramecia

Summary Phage-like particles from kappa of stock 562 of Paramecium aurelia have been isolated by CsCl density gradient centrifugation. Analyses show that the particles contain about 1.6×10¹⁶g DNA and 2.0×10^-16g protein. Their buoyant density is approximately 1.47. DNA from the particles has a buoyant density very close to that of whole kappa DNA. The presence of DNA in the particles has been confirmed by a cytochemical technique. The results support the conclusion that kappa contains a bacteriophage.     

Novel evolutionary insights on the interactions of the Holosporales (Alphaproteobacteria) with eukaryotic hosts from comparative genomics

Holosporales are an alphaproteobacterial order engaging in obligate and complex associations with eukaryotes, in particular protists. The functional and evolutionary features of those interactions are still largely undisclosed. Here, we sequenced the genomes of two members of the species Bealeia paramacronuclearis (Holosporales, Holosporaceae) intracellularly associated with the ciliate protist Paramecium, which resulted in high correspondence. Consistent with the short-branched early-divergent phylogenetic position, Bealeia presents a larger functional repertoire than other Holosporaceae, comparable to those of other Holosporales families, particularly for energy metabolism and motility. Our analyses indicate that different Holosporales likely experienced at least partly autonomous genome reduction and adaptation to host interactions, for example regarding dependence on host biotin driven by multiple independent horizontal acquisitions of transporters. Among Alphaproteobacteria, this is reminiscent of the convergently evolved Rickettsiales, which however appear more diverse, possibly due to a probably more ancient origin. We identified in Bealeia and other Holosporales the plasmid-encoded putative genetic determinants of R-bodies, which may be involved in a killer trait towards symbiont-free hosts. While it is not clear whether these genes are ancestral or recently horizontally acquired, an intriguing and peculiar role of R-bodies is suggested in the evolution of the interactions of multiple Holosporales with their hosts.     

Aerobic Respiration of Kappa Particles from Paramecium aurelia,Stock 51*

SYNOPSIS. Kappa particles from killer cultures of stock 51 Paramecium aurelia were purified and their respiration measured polarographically. The slight bacterial contaminations in the kappa preparations were not significant. Freshly collected kappa in dilute buffer at room temperature had an endogenous Q_O2 of 17.0 ± 1.6 μl/mg dry weight/hr (mean ± standard error). The Q_O2 decayed 50% in 5 hr. Among the sugars tested only glucose and sucrose increased the respiratory rate of kappa. The di- and tri-carboxylates of the tricarboxylic acid cycle stimulated the respiration of kappa. KCN, CO and 2-heptyl-4-hydroxyquinoline N-oxide (HOQNO) inhibited respiration. These findings ensure an organismic status for kappa and justify the belief that it is bacterial in origin.     

A comparison of the refractile bodies (R-bodies) of certain bacteria—II. Effects of pH on the structure

The ‘R-bodies’ of Caedibacter taeniospiralis, an endosymbiont of Paramecium tetraurelia and Pseudomonas avenae, have been examined under a wide range of pH. They both unroll over a small range, but in both cases the process does not always proceed to completion. Acid pHs cause both to unroll. All the R-bodies of P. avenae at high pHs are wound which is not the case with Caedibacter. There are differences between the two bodies in the details of their unrolling. Caedibacter taenospiralis unwinds from the inside, whereas P. avenae unwinds from the outside. Detailed study of the unwound ribbon shows a difference in terms of roughness and smoothness of the two surfaces.     

Studies on R-bodies of Pseudomonas Avenae

Utilizing the techniques of thin sectioning, negative staining and scanning electron microscopy R-bodies have been observed to occur in pairs in different relationships to each other. A very large proportion of the volume of the bacterial cell may be occupied by R-bodies with as many as three R-bodies being intertwined.     

The localization of the toxins produced by R-body containing bacteria

Immunogold labelling techniques allied with electron microscopy have shown that the toxin of Pseudomonas avenae which is isolated from the growth medium localizes on the cell surface of the bacterium. Antisera against P. avenae R-bodies, however, cross-react with the specific R-body structure in a cell.     

Towards an ecological understanding of the killer trait – A reproducible protocol for testing its impact on freshwater ciliates

Paramecium strains with the ability to kill other paramecia often harbour intracellular bacteria belonging to the genera Caedibacter or Caedimonas. Central structures of this killer trait are refractile bodies (R-bodies) produced by the endosymbionts. Once ingested by a sensitive Paramecium, R-bodies presumably act as delivery system for an unidentified toxin which causes the death of endosymbiont-free paramecia while those infected gain resistance from their symbionts. The killer trait is therefore considered as competitive advantage for the hosts of R-body producers. While its effectiveness against paramecia is well documented, the effects on other aquatic ciliates are much less studied.In order to address the broadness of the killer trait, a reproducible killer test assay considering the effects on predatory ciliates (Climacostomum virens and Dileptus jonesi) as well as potential bacterivorous Paramecium competitors (Dexiostoma campyla, Euplotes aediculatus, Euplotes woodruffi, and Spirostomum teres) as possibly susceptible species was established. All used organisms were molecularly characterized to increase traceability and reproducibility. The absence of any lethal effects in both predators and competitors after exposure to killer paramecia strongly suggests a narrow action range for the killer trait. Thus, R-body producing bacteria provide their host with a complex, costly strategy to outcompete symbiont-free congeners only.     

R-bodies inPseudomonas aeruginosa strain 44T1

For the first time R-bodies are described in a new strain 44T1 ofPseudomonas aeruginosa. Its size was measured as being 0.22 to 0.37 m of width per 0.27 to 0.41 m of length and 5 to 9 spiral turns about 16 nm. These structures are similar to previously observed in bacteria and are related with physiological state of bacteria in minimal conditions of growth.     

Experimental Microbial Populations Thirty-five Years Later: The Influence of Food on the Symbiosis of Paramecium aurelia Syngen 4, 51.7

Changes in the nutrition of Paramecium aurelia affect its ability to serve as host for the bacteroid parasite, kappa, and the presence or absence of kappa affects its ability to grow in axenic culture. Loss of kappa, tested by the presence or absence of killer reaction, occurred in cultures of P. aurelia growing at a reduced division rate on autoclaved Enterobacter aerogenes in suspensions of lettuce and yeast autolysate 14–17 days after they had been rendered bacteria-free by washing. Killer Paramecium sterilized of bacteria by treatment with an antibiotic mixture of penicillin-G and streptomycin in combination with a nonbacterial nonliving culture medium, lost the ability to kill after from 6 to 48 hours in the sterilizing medium. The ciliates from which kappa had been lost during exposure to antibiotics could be transferred immediately and maintained in axenic culture, but those washed free of bacteria could not be maintained axenically until kappa had been lost during cultivation in a medium containing killed bacteria. It is suggested that a knowledge of the nutritional requirements of symbiotic microorganisms is essential for understanding the ecological aspects of eutrophication of aquatic environments.     

Tracing the role of R-bodies in the killer trait: Absence of toxicity of R-body producing recombinant E. coli on paramecia

R-bodies are coiled proteinaceous ribbons produced by Paramecium endosymbionts belonging to the genus Caedibacter. These intracellular bacteria confer upon their hosts a phenomenon called the killer trait. It is the ability to kill symbiont-free competitors called sensitives. The R-body is the crucial element of this process, but despite many efforts, the actual role of R-bodies in killing sensitive paramecia is still not satisfactory clarified. The open question is whether the R-body acts as transmitter for a yet unidentified toxin or whether it directly kills sensitive paramecia having intrinsic cytotoxic effects. In the present study, this problem is addressed by heterologous expression of Caedibacter taeniospiralis R-body in Escherichia coli followed by a detailed analysis of its potential intrinsic toxic effect on feeding sensitive Paramecium tetraurelia. Using this approach, we can exclude any eventual effects of additional, unidentified factors produced by C. taeniospiralis and thus observe the impact of the recombinant R-body itself. No cytotoxic effects of recombinant R-bodies were detected following this approach, strengthening the hypothesis that R-bodies act as releasing system for an unidentified C. taeniospiralis toxin.     

CARRAGEENANS BIOSYNTHESIZED BY CARPOSPOROPHYTES OF RED SEAWEEDS GIGARTINA SKOTTSBERGII (GIGARTINACEAE) AND GYMNOGONGRUS TORULOSUS (PHYLLOPHORACEAE)1

Carrageenans biosynthesized by gametophytic and tetrasporic plants of seaweeds belonging to the Gigartinaceae and Phyllophoraceae are different: gametophytes produce carrageenans of the kappa family, whereas lambda‐carrageenans are extracted from tetrasporophytes. For Gigartina skottsbergii Setchell and Gardner and Gymnogongrus torulosus Hooker et Harvey, mature cystocarps were isolated and carrageenans were extracted. Structural determination by methylation analysis, Fourier transform infrared spectroscopy, and ¹³C‐NMR spectroscopy showed that they were kappa/iota‐carrageenans. For the extract obtained from cystocarps of Gigartina skottsbergii with water at room temperature, the ratio kappa:iota was 1:0.30 and at 90° C was 1:0.43; significant amounts of precursors were also present. The extract obtained from cystocarps of Gymnogongrus torulosus at 90° C showed prevalence of iota‐carrageenans (ratio kappa:iota 1:1.21). These extracts are similar to the polysaccharides produced by gametophytes of these seaweeds. For Gigartina skottsbergii, it was possible to separate the pericarpic tissue from the carposporophyte. Thus, they were extracted separately, and the carrageenans isolated were studied as described before, obtaining similar conclusions. These results clearly show that whereas the carposporophytes are located inside the cystocarp, they produce carrageenans of the kappa family despite of being diploid cells.     

Environmental influences on bovine kappa-casein: reduction and conversion to fibrillar (amyloid) structures

The caseins of milk form a unique calcium–phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of -casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the -casein (90°C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high–molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem. 17: 73–84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-). Surprisingly, at only 37°C, the RCM--casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37°C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM--caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turnsheet motif in -casein (residues 15–65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM--casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland.     

Duplication of J κ genes within genus Rattus

We have isolated a region containing the immunoglobulin kappa chain joining segments from a liver DNA library of the Australian rat Rattus villosissimus, and determined its nucleotide sequence. While the laboratory rat (Rattus norvegicus) had previously been shown to contain three recently duplicated copies of J 2, R. villosissimus has only two. Furthermore, all three copies of J 2 in R. norvegicus share an 11 by deletion in their 5 flanking regions which is not evident in either copy of J 2 in R. villosissimus. This suggests that the initial duplication events occurred separately in the two lineages, and were followed by a second duplication in R. norvegicus, all three duplications having occurred within the last 6–12 million years (although more complicated schemes involving gene conversion events cannot be excluded). These results indicate that there is a high degree of plasticity in this region of the genome, and that selective forces must exist which have maintained the number of expressible J segments in humans (5) and rodents (4–6) within their narrow range.     

The role of wing geometric morphometrics in the identification of sandflies within the subgenus Lutzomyia

The Lutzomyia subgenus (Diptera: Psychodidae) includes sibling species with morphologically indistinguishable females. The aims of this study were to analyse variations in the size and shape of wings of species within the Lutzomyia subgenus and to assess whether these analyses might be useful in their identification. Wings (n = 733) of 18 species deposited in Brazilian collections were analysed by geometric morphometrics, using other genera and subgenera as outgroups. Shape variation was summarized in multivariate analyses and differences in wing size among species were tested by analysis of variance. The results showed significant variation in the sizes and shapes of wings of different Lutzomyia species. Two clusters within the Lutzomyia subgenus were distinguished in analyses of both males and females. In Cluster 1 (Lutzomyia ischnacantha, Lutzomyia cavernicola, Lutzomyia almerioi, Lutzomyia forattinii, Lutzomyia renei and Lutzomyia battistinii), scores for correct reclassification were high (females, kappa = 0.91; males, kappa = 0.90), whereas in Cluster 2 (Lutzomyia alencari, Lutzomyia ischyracantha, Lutzomyia cruzi, Lutzomyia longipalpis, Lutzomyia gaminarai and Lutzomyia lichyi), scores for correct reclassification were low (females, kappa = 0.42; males, kappa = 0.48). Wing geometry was useful in the identification of some species of the Lutzomyia subgenus, but did not allow the identification of sibling species such as L. longipalpis and L. cruzi.     

Recombination between kappa chain genetic markers in the mouse

In this study we report the first instance of recombination between kappa chain genetic markers in the mouse. The recombination frequency, 0.45% (95% limits, 0.12–1.61), is similar to that previously found for recombination between the kappa chain locus and the Lyt-2, 3 locus (0.3%, 95% limits, 0.05–1.6), but is relatively low in comparison with that found at the heavy chain locus (0.41–5.4%). Lyt-2, 3-typing of the recombinants permits a partial ordering of the kappa chain and Lyt-2, 3 loci as (Lyt-2, 3, Igk-Ef1) - Igk-Ef2. Light chains controlled by the two kappa markers include the V_k-(ser) subgroup (controlled by Igk-Ef1) and V_k–1 (controlled by Igk-Ef2). One of the recombinants has been recovered in a homozygous state (NAK) and should be suitable for V _k gene mapping studies.Abbreviations C complement - C_H constant region of the Ig heavy chain - CI cytotoxicity index - DNP dinitrophenyl - FMF flow microfluorimetry - IEF isoelectric focusing - IF immunofluorescence - Ig immunoglobulin - KLH keyhole limpet hemocyanin - V_H variable region of the Ig heavy chain - V_k variable region of the Ig kappa chain - V-region variable region     

Human immunoglobulin variable region genes: V κ polymorphisms suggest variation in V-gene repertoires

The present study characterizes four potentially informative polymorphic bands of 5.2, 2.3, 1.9, and 1.2 kb, detected by Southern blot hybridization of Eco RI digests of human DNA using HK101/80 (an immunoglobulin V I probe). These restriction fragment length polymorphisms (RFLP) show Mendelian segregation and they are linked to each other and to Km(1), the allotypic marker on the kappa constant region. There is strong linkage disequilibrium between the 2.3 and 1.2 kb polymorphisms. A 0.7 kb Pst I polymorphic band and a 2.9 kb Sac I polymorphic band were also identified; the latter may reflect a region of tandem repeats in the V region. No bands representing the alternative forms of any of the polymorphic restriction sites were identified. This implies either that genes are missing from the V repertoire or that such bands are hidden because of comigration of fragments due to conservation of restriction sites. Evidence for comigration of fragments was obtained from independent V clones and suggests that dark bands on Southern blots of Pst I digests must often represent several superimposed genes which have conserved restriction sites. The demonstration of RFLP within the V region provides circumstantial evidence for polymorphic variation in the repertoire of V structural genes. The RFLP reported here should be useful as genetic markers in future studies on the immune response and disease susceptibility in man.     

Calcium-induced associations of the caseins: Thermodynamic linkage of calcium binding to colloidal stability of casein micelles

The caseins occur in milk as colloidal complexes of protein aggregates, calcium, and inorganic phosphate. As determined by electron microscopy, these particles are spherical and have approximately a 650 Å radius (casein micelles). In the absence of calcium, the protein aggregates themselves (submicelles) have been shown to result from mainly hydrophobic interactions. The fractional concentration of stable colloidal casein micelles can be obtained in a calcium caseinate solution by centrifugation at 1500g. Thus, the amount of stable colloid present with varying Ca²⁺ concentrations can be determined and then analyzed by application of equations derived from Wyman's Thermodynamic Linkage Theory. Ca²⁺-induced colloid stability profiles were obtained experimentally for model micelles consisting of only _s1- (a calcium insoluble casein) and the stabilizing protein -casein, eliminating the complications arising from - and minor casein forms. Two distinct genetic variants _s1-A andB were used. Analysis of _s1-A colloid stability profiles yielded a precipitation (salting-out) constantk ₁, as well as colloid stability (salting-in) parameterk ₂. No variations ofk ₁ ork ₂ were found with increasing amounts of -casein. From the variation of the amount of colloidal casein capable of being stabilized vs. amount of added -casein an association constant of 4 L/g could be calculated for the complexation of _s1-A and -casein. For the _s1-B and -casein micelles, an additional Ca²⁺-dependent colloidal destabilization parameter,k ₃, was added to the existingk ₁ andk ₂ parameters in order to fully describe this more complex system. Furthermore, the value ofk ₃ decreased with increasing concentration of -casein. These results were analyzed with respect to the specific deletion which occurs in _s1-caseinA in order to determine the sites responsible for these Ca²⁺-induced quaternary structural effects.     

Genetic polymorphism at the κ chain locus in mice: Comparisons of restriction enzyme hybridization fragments of variable and constant region genes

Variable (V) and constant (C) region genes of the mouse kappa light chain have been compared in inbred strains and in geographically isolated or genetically separated populations of mice by Southern blot analysis of endonuclease-restricted germline DNA. In most cases, the C gene is found on a single restriction fragment while the V genes of the V19 and V21 groups are each found on several (6–18) fragments. The restriction fragment (RF) patterns of V19 and V21 groups are both polymorphic when compared among inbred mouse strains. Southern blot patterns of V21 and V19 of inbred strains are also found among some geographically isolated populations of mice, suggesting that inbred strains acquired kappa loci from different subspecies. Some populations of geographical isolates show V21, V19, and C contexts similar to inbred mice while more distantly related species within the genus Mus and laboratory rats show no apparent similarity in context to inbred strains. Variable region genes determining the RF patterns of V19 and V21 appear to be linked to each other and to the C and Lyt-3 loci.     

Linked genetic markers of the rabbit κ light chain are not linked to the Tcr β chain genes

In order to investigate linkage, we used serum allotypes of the two rabbit C isotypes and restriction fragment length polymorphisms (RFLPs) of the genes for V , C , and T-cell receptor C . The inheritance of these genetic markers was studied through backcross and F₂ matings. Southern analysis and hybridization of genomic DNA with a C probe detected a 5 kb Pst I fragment linked to expression of the K2bas1 allotype and the presence of the 1b ^bas gene and a 6.6 kb Pst I fragment linked to the expression of the K1b9 allotype, the presence of the 2 ^bas2 gene and lack of expression of the K2bas1 allotype. A V probe detected a 1.3 kb Eco RI fragment linked to the presence of the 1b ^bas gene and expression of the K2bas1 allotype. In contrast, the 9 or 14 kb Eco RI RFLP (C ^a or C ^b) detected with a Tcr chain probe segregated independently from C allotypes and RFLPs. It has previously been found that C and C are also unlinked in man, whereas in the mouse they are linked at a distance of 8 centimorgans.