R-Bodies in newly isolated free-living hydrogen-oxidizing bacteria

R-Bodies have been found in a recently isolated pseudomonas-like free-living hydrogen oxidizing bacterium. Their isolation, fine structure and chemical composition are described and compared with the R-bodies from the kappa particles (Caedobacter), obligate endosymbionts of Paramecium aurelia. The 2K 1 R-bodies exhibited essential characteristics of the kappa R-bodies; however, their size and some other structural aspects proved that they represent a new type of R-bodies. The presence of phage tail-like particles in cells induced with Mitomycin C is in favour of the hypothesis that the R-bodies might be coded by defective prophages, or by extrachromosomal elements.     

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.     

A comparison of the refractile bodies (R-bodies) of certain bacteria—III. Nucleotide sequence homologies and R-body function

The R-bodies found in certain bacteria symbiotic in paramecia and in some free living species have been examined directly to see if they kill or affect other cells following microinjection and on addition to the media. No effects were seen on several cell types. Some symbionts when added to the media killed but not when they were microinjected. Cytoplasm and soluble fractions from certain paramecia killed when microinjected into paramecia but not if the latter contained other symbionts. The role of the R-body and its association with toxic effects is still not clear.Studies of the base composition and nucleic acid homologies of the bacteria producing R-bodies shows there is little genetic relationship between them, suggesting that the sequences responsible for R-body synthesis may have been mobile and changed during evolution.     

Trichocyst Ribbons of a Cryptomonads Are Constituted of Homologs of R-Body Proteins Produced by the Intracellular Parasitic Bacterium of Paramecium

Trichocysts are ejectile organelles found in cryptomonads, dinoflagellates, and peniculine ciliates. The fine structure of trichocysts differs considerably among lineages, and their evolutionary relationships are unclear. The biochemical makeup of the trichocyst constituents has been studied in the ciliate Paramecium, but there have been no investigations of cryptomonads and dinoflagellates. Furthermore, morphological similarity between the contents of cryptomonad trichocysts and the R-bodies of the endosymbiotic bacteria of Paramecium has been reported. In this study, we identified the proteins of the trichocyst constituents in a red cryptomonad, Pyrenomonas helgolandii, and found their closest relationships to be with rebB that comprises the R-bodies of Caedibacter taeniospiralis (gammaproteobacteria), which is an endosymbiont of Paramecium. In addition, the biochemical makeups of the trichocysts are entirely different between cryptomonads and peniculine ciliates, and therefore, cryptomonad trichocysts have an evolutionary origin independent from the peniculine ciliate trichocysts.     

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.     

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.     

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.     

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.     

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.     

Frequency and biodiversity of symbionts in representatives of the main classes of Ciliophora

Representatives of all classes of Ciliophora have been studied for the detection and investigation of both prokaryotic and eukaryotic (not algal) endo- (EnS) and ectosymbionts (EcS). Different methods including transmission electron microscopy (TEM) and fluorescence in situ hybridisation (FISH) have been used. Apparently, the capability of keeping symbionts varies among the different ciliate groups as it generally is the case in different protist taxa. Most of the prokaryotic EnSs detected belong to Alphaproteobacteria. Holospora or Holospora-like infectious bacteria of this group were found in representatives of Heterotrichea, Armophorea, Phyllopharyngea, Prostomatea and mainly of Oligohymenophorea. Bacteria associated with bacteriophages were found in species of Heterotrichea and Oligohymenophorea. This holds true also for bacteria with R-bodies. A quite rare type of EnS - motile bacteria - was found in ciliates of the same two classes as well, either in the cytoplasm (Heterotrichea) or in the macronucleus and its perinuclear space (Oligohymenophorea). EcSs are more common in Heterotrichea, Armophorea and Plagiopylea, but were never found in other groups. Among the eukaryotic EnSs of ciliates, very few representatives of Microsporidia and Trypanosomatidae were recorded. In conclusion, heterotrichs and oligohymenophoreans are the most promising groups of Ciliophora for the investigation of symbiosis.     

Ejectisins: tough and tiny polypeptides are a major component of cryptophycean ejectisomes

Fragments of discharged ejectisomes were isolated from two Cryptomonas and a Chroomonas species by detergent treatment followed by Percoll density gradient centrifugation. The fragments withstand high concentrated detergent solutions, reducing agents and freeze-thawing. Disintegration was achieved in 6 M guanidine hydrochloride. Reassembly into long, filamentous, ejectisome-like structures occurred after dialysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the polypeptide patterns of isolated ejectisome fragments and of reconstituted ejectisome-like structures were dominated by polypeptides with relative molecular weights of approximately 6 kDa. The polypeptides were not glycosylated and did not cross-react with antisera directed against recombinant Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis. A polyclonal antiserum directed against reconstituted, ejectisome-like filaments cross-reacted with the 6-kDa polypeptides and immunolabeled extruded ejectisome filaments. Twenty amino acid residues, obtained by N-terminal amino acid sequence analysis, matched to polypeptide sequences deduced from cDNA sequences of the cryptophyte Guillardia theta. The term “ejectisins” is introduced for the 6-kDa polypeptides which represent a major component of cryptophycean ejectisomes.     

The Toxic Symbiont Caedibacter caryophila in the Cytoplasm of Paramecium novaurelia

Endosymbiotic bacteria were observed to inhabit the cytoplasm of the freshwater ciliateParamecium novaurelia. Transmission electron microscopy and toxicity tests with sensitive paramecia showed that the endosymbionts belong to the genusCaedibacter. The bacteria conferred a killer trait to their host paramecia. The production of a proteinaceous inclusion body (“R-body”) in the bacterial cell makes them toxic to other paramecia after they become enclosed in food vacuoles. R-bodies ofCaedibacter sp were associated with phages, which are known in most otherCaedibacter species to code for the R-body proteins. The killer-effect ofP. novaurelia on sensitiveP. caudatum strains was of the “paralysis” type, which is a characteristic of the symbiont speciesCaedibacter caryophila. Until nowC. caryophila was known to inhabit the macronucleus ofParamecium caudatum only. Sequencing of the 16S rRNA-gene proved thatCaedibacter sp from the cytoplasm ofP. novaurelia belongs to the speciesC. caryophila as well. The rDNA-sequence of 1695 bp length differed in a total of only 1 bp from the corresponding gene inC. caryophila from the macronucleus ofP. caudatum. The results indicate that the infection of specific host cell compartments may depend on host genes, but not on different traits of the infecting symbiont species. The occurrence of killer and sensitive paramecia strains together in one pond is discussed with respect to the competitive advantage of the killer trait.     

Phylogenetic relationships among endosymbiotic R-body producer: Bacteria providing their host the killer trait

R-body producing bacterial endosymbionts of Paramecium spp. transform their hosts into “killer” paramecia and provide them a selective advantage. This killer trait is connected to the presence of R-bodies, which are peculiar, tightly coiled protein ribbons capable of rapid unrolling. Based mainly on those two characteristics the respective obligate intracellular bacteria have been comprised in the genus Caedibacter and additional traits such as host species, subcellular localization, and R-body dimensions and mode of unrolling were used for species discrimination. Previous studies applying the full-cycle rRNA approach demonstrated the polyphyly of this assemblage. Following this approach, we obtained new sequences and in situ hybridizations for five strains of Caedibacter taeniospiralis and four strains associated to Caedibacter varicaedens and Caedibacter caryophilus. Detailed phylogenetic reconstructions confirm the association of C. taeniospiralis to Fastidiosibacteraceae and to Holosporales in case of the others. Therefore, we critically revise the taxonomy of the latter group. The high 16S rRNA gene sequence similarity among the type strains of Caedibacter varicaedens and C. caryophilus indicate that they should be classified within a single species for which we propose Caedimonas varicaedens comb. nov. owing to the priority of Caedibacter varicaedens. Moreover, we propose to establish the new family Caedimonadaceae fam. nov. to encompass Caedimonas varicaedens, “Ca. Paracaedimonas acanthamoebae” comb. nov. and “Ca. Nucleicultrix amoebiphila” within the order Holosporales.     

Genome of the R-body producing marine alphaproteobacterium Labrenzia alexandrii type strain (DFL-11T)

Labrenzia alexandrii Biebl et al. 2007 is a marine member of the family Rhodobacteraceae in the order Rhodobacterales, which has thus far only partially been characterized at the genome level. The bacterium is of interest because it lives in close association with the toxic dinoflagellate Alexandrium lusitanicum. Ultrastructural analysis reveals R-bodies within the bacterial cells, which are primarily known from obligate endosymbionts that trigger “killing traits” in ciliates (Paramecium spp.). Genomic traits of L. alexandrii DFL-11^T are in accordance with these findings, as they include the reb genes putatively involved in R-body synthesis. Analysis of the two extrachromosomal elements suggests a role in heavy-metal resistance and exopolysaccharide formation, respectively. The 5,461,856 bp long genome with its 5,071 protein-coding and 73 RNA genes consists of one chromosome and two plasmids, and has been sequenced in the context of the Marine Microbial Initiative.     

Factors associated with birth rate and live birth rate in multi-male breeding groups of rhesus monkeys

Reproductive records of 284 female rhesus monkeys housed in six multimale corrals at the California Primate Research Center were examined for the birth seasons 1977–1982 to determine possible associations between the probability of birth or live birth and female age, parity, origin, parturition in the previous season, infant birth date, and infant birth date in previous season. Multiple logistic regression analysis was used to identify and quantitate the effects of factors on the probability of birth or live birth, while controlling for the possibly confounding effects of other factors in the model. Females who had infants early in the previous season were 2.5 times as likely to give birth as those who had infants late in the previous season. Females with two or three previous births were 2.1 times as likely to give birth, and those with four or five previous births were 6.7 times as likely to give birth as were females with no or one previous birth. Controlling for other factors (age, parity, and timing of birth in the previous season), corralborn females were 3.3 times as likely to give birth as either wild-caught or domestic-born monkeys not native to the corrals. Domestic-born females who were not corral natives were 0.3 times as likely to have live births as wild-caught females. Births late in the season were 1.8 times as likely to result in live infants as births early in the season.     

Biochemical mechanism of combined effect of ethambutol and sparfloxacin against Mycobacterium smegmatis

M. smegmatis cells grown in the presence of combination of ethambutol (EMB) and sparfloxacin (SPX) had decreased level of total cellular lipids as compared to control as well as cells grown in the presence of sub-inhibitory concentration (MIC50) of individual drugs. Amongst various phospholipids analyzed, maximum decrease was observed in the content of phosphatidylinositolmannosides (PIMs) of the cells grown in combination of EMB and SPX. In contrast, the subcellular distribution of phospholipids revealed a significant increase in PIMs content of both cell wall and cell membrane of the cells grown in the presence of combination of drugs as compared to control as well as individual drugs. Mycolic acids of M. smegmatis cells were found to be main targets as combination of drugs resulted in significant decrease in total cellular as well as cell wall mycolic acids as compared to control and individual drugs. Changed lipid composition of M. smegmatis cells grown in the presence of MIC50 of EMB, SPX and combination resulted in significant surface changes as was evident from decreased limiting fluorescence (Fmax) intensity of 1-anilinonaphthalene-8-sulfonate (ANS). Thus, the results of this study suggested that ethambutol and sparfloxacin in combination exerted their antimycobacterial effect principally due to their action on phosphatidylinositolmannosides (PIMs) and mycolic acids, which form the permeability barrier of mycobacteria.     

Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA.

A nonadecanucleotide has been used both as a site specific mutagen to introduce a T leads to A transversion mutation in the human beta-globin gene cloned in pBR322 as well as a probe to screen transformed colonies for the desired mutant. The specificity of the oligonucleotide as a mutagen and as a hybridization probe provide a general method for producing site specific mutations in DNA cloned in plasmid vectors such as pBR322.     

Natural solution to antibiotic resistance: bacteriophages ‘The Living Drugs’

Antibiotics have been a panacea in animal husbandry as well as in human therapy for decades. The huge amount of antibiotics used to induce the growth and protect the health of farm animals has lead to the evolution of bacteria that are resistant to the drug’s effects. Today, many researchers are working with bacteriophages (phages) as an alternative to antibiotics in the control of pathogens for human therapy as well as prevention, biocontrol, and therapy in animal agriculture. Phage therapy and biocontrol have yet to fulfill their promise or potential, largely due to several key obstacles to their performance. Several suggestions are shared in order to point a direction for overcoming common obstacles in applied phage technology. The key to successful use of phages in modern scientific, farm, food processing and clinical applications is to understand the common obstacles as well as best practices and to develop answers that work in harmony with nature.     

Comet assay: a reliable tool for the assessment of DNA damage in different models

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans. IITR Communication No. 2656     

Protective effects of green tea catechins on alveolar macrophages against bacterial infections

Bacterial pneumonia in immunocompromised patients as well as elderly persons often becomes a life threatening disease, even when effective antibiotics are used extensively. In addition, the appearance of antibiotic-resistant bacteria in medical facilities as well as in patients requires another approach to treat such patients besides treatment with antibiotics. In this regard, green tea catechins, such as epigallocatechin gallate (EGCg), may be one of the potential agents for such purpose due to its possible potential immunomodulatory as well as antimicrobial activity. The studies by us showed that EGCg enhanced the in vitro resistance of alveolar macrophages to Legionella pneumophila infection by selective immunomodulatory effects on cytokine formation. Furthermore, the tobacco smoking-induced impairment of alveolar macrophages regarding antibacterial as well as immune activity was also recovered by EGCg treatment. These results indicate that EGCg may be a possible potential immunotherapeutic agent against respiratory infections in immunocompromised patients, such as heavy smokers.