Paramecium caudatum acquires heat-shock resistance in ciliary movement by infection with the endonuclear symbiotic bacterium Holospora obtusa

Holospora obtusa is a macronucleus-specific bacterium of the ciliate Paramecium caudatum. Three types of P. caudatum cells (H. obtusa-free cells, cells bearing the reproductive form of H. obtusa and cells bearing the predominantly infectious form of H. obtusa) cultured at 25 degrees C were transferred to 4, 10, 25, 35 and 40 degrees C and their swimming velocities were measured by taking photomicrographs with two-second exposures. The H. obtusa-free cells almost ceased swimming at both 4 and 40 degrees C, while cells bearing the reproductive form and those bearing the predominantly infectious form actively swam even at these temperatures. These results show that the host cell can acquire heat-shock resistance when infected by H. obtusa in the macronucleus. This is the first evidence to show that the endonuclear symbiont Holospora contributes to maintain the ciliary movement of the host even at temperatures unsuitable for the host growth.     

Fate of the 63-kDa periplasmic protein of the infectious form of the endonuclear symbiotic bacterium Holospora obtusa during the infection process

Holospora obtusa is a macronucleus-specific endosymbiotic bacterium of the ciliate Paramecium caudatum. We report the secretion of a 63-kDa periplasmic protein of an infectious form of the bacterium into the macronucleus of its host. Indirect immunofluorescence microscopy with five monoclonal antibodies against the 63-kDa protein demonstrated that, soon after the bacterial invasion into the host macronucleus, the protein was detected in the infected macronucleus and that levels of the protein increased dramatically within one day of infection. The use of inhibitors for host and bacterial protein synthesis illustrated that, in early infection of H. obtusa, not only the pre-existing but also a newly synthesized 63-kDa protein was secreted into the host macronucleus. A partial amino acid sequence of the protein was determined, and a gene encoding the 63-kDa protein was cloned. The deduced amino acid sequence shows that this protein is a novel protein.     

Effects of antibiotics on the early infection process of a macronuclear endosymbiotic bacterium Holospora obtusa of Paramecium caudatum

We examined the effects of antibiotics involved in bacterial DNA, RNA and protein synthesis and host protein synthesis on the early infection process of the bacterium Holospora obtusa, a macronucleus-specific symbiont of the ciliate Paramecium caudatum. Infection of the host macronucleus by the bacterium was not inhibited by mitomycin C, rifampicin and chloramphenicol. However, ingestion of the bacterium into the host digestive vacuoles and escape of the bacterium from the vacuoles to the host cytoplasm were significantly arrested with emetine. The results suggest that newly synthesized host proteins play an important role in the early infection process.     

The Release of Holospora obtusa from Paramecium caudatum Observed with a New Device for Extended In Vivo Microscopy

The macronudeus-specilic bacterium Holospora obtusa is released from its host cell Paramecium caudatum generally after a preceding cell division. Before division, most infectious forms of the bacteria can be found in a connecting strand of the dividing macronucleus. This connecting strand is deformed and finally expelled as a whole from the daughter cells. This process was observed and photographed with a simple device allowing observations for a long lime under a microscope without damage to the living cells. Key words. Bacterium, cytoproct, defecation, infection, macronucleus, symbiosis.     

Micronucleus‐Specific Bacterium Holospora elegans Irreversibly Enhances Stress Gene Expression of the Host Paramecium caudatum

ABSTRACT. The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear‐specific symbiont Holospora obtusa survived better than symbiont‐free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25 °C, a usual growth temperature. We report herein that paramecia bearing the micronuclear‐specific symbiont Holospora elegans also acquire the heat‐shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock‐resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25 °C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat‐shock resistance.     

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum

Summary. Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria. Correspondence and reprints: Department of Cytology and Histology, Faculty of Biology and Soil Sciences, Saint Petersburg State University, Universitetskaya naberezhnaya 7/9, 199034 Saint Petersburg, Russia.     

Obtaining and characterization of “Holospora curviuscula” and Holospora obtusa, bacterial symbionts of the macronuclei of Paramecium bursaria and Paramecium caudatum

The symbionts of the macronuclei of Paramecium bursaria and P. caudatum, “Holospora curviuscula” 02AZ16 and H. obtusa 88Ti, respectively, were obtained and investigated. The 16S rDNA nucleotide sequences of “Holospora curviuscula” were obtained for the first time. The differences in 16S rDNA (3.4%) suggest their classification within the genus Holospora. Molecular phylogenetic analysis of the symbionts revealed that these intranuclear symbionts of the ciliates belonged to the order Rickettsiales, forming within a compact cluster of related species.     

Co-infection of the macronucleus of Paramecium caudatum by free-living bacteria together with the infectious Holospora obtusa

Infection experiments were performed incubating Paramecium caudatum with non-infectious free-living bacteria or weakly infectious intracellular bacteria together with the infectious Holospora obtusa. Two of four non-infectious free-living bacteria (Enterobacter aerogenes and Klebsiella pneumoniae) were found to get into the nuclei when added to Paramecium together with H. obtusa. The endonuclear bacterium Nonospora macronucleata that is weakly infectious by itself increases its infectivity when presented together with the infectious holosporas. The results provide evidence that H. obtusa may facilitate entry of other, non-infectious bacteria into the nuclei of Paramecium.     

The endosymbiotic bacterium Holospora obtusa enhances heat-shock gene expression of the host Paramecium caudatum

The bacterium Holospora obtusa is a macronuclear-specific symbiont of the ciliate Paramecium caudatum. H. obtusa-bearing paramecia could survive even after the cells were quickly heated from 25 degrees C to 35 degrees C. To determine whether infection with H. obtusa confers heat shock resistance on its host, we isolated genes homologous to the heat shock protein genes hsp60 and hsp70 from P. caudatum. The deduced amino acid sequences of both cDNAs were highly homologous to hsp family sequences from other eukaryotes. Competitive PCR showed that H. obtusa-free paramecia expressed only trace amounts of hsp60 and hsp70 mRNA at 25 degrees C, but that expression of hsp70 was enhanced immediately after the cells were transferred to 35 degrees C. H. obtusa-bearing paramecia expressed high levels of hsp7O mRNA even at 25 degrees C and the level was further enhanced when the cells were incubated at 35 degrees C. In contrast, the expression pattern of hsp60 mRNA was the same in H. obtusa-bearing as in H. obtusa-free paramecia. These results indicate that infection with its endosymbiont can confer a heat-shock resistant nature on its host cells.     

“Candidatus Paraholospora nucleivisitans”, an intracellular bacterium in Paramecium sexaurelia shuttles between the cytoplasm and the nucleus of its host

An intracellular bacterium was discovered in two isolates of Paramecium sexaurelia from an aquarium with tropical fish in Münster (Germany) and from a pond in the Wilhelma zoological–botanical garden, Stuttgart (Germany). The bacteria were regularly observed in the cytoplasm of the host, but on some occasions they were found in the macronucleus of the host cell. In these cases, only a few, if any, bacteria were observed remaining in the cytoplasm. The bacterium was not infectious to P. sexaurelia or other species of Paramecium and appeared to be an obligate intracellular bacterium, while bacteria-free host cells were completely viable. The fluorescence in situ hybridisation (FISH) and comparative 16SrDNA sequence analyses showed that the bacterium belonged to a new genus, and was most closely, yet quite distantly, related to Holospora obtusa. In spite of this relationship, the new bacteria differed from Holospora by at least two biological features. Whereas all Holospora species reside exclusively in the nuclei of various species of Paramecium and show a life cycle with a morphologically distinct infectious form, for the new bacterium no infectious form and no life cycle have been observed. For the new bacterium, the name Candidatus Paraholospora nucleivisitans is suggested. The host P. sexaurelia is usually known from tropical and subtropical areas and is not a species typically found in Germany and central Europe. Possibly, it had been taken to Germany with fish or plants from tropical or subtropical waters. Candidatus Paraholospora nucleivisitans may therefore be regarded as an intracellular neobacterium for Germany.     

Structure and Expression of a GroE-homologous Operon of a Macronucleus-Specific Symbiont Holospora obtusa of the Ciliate Paramecium caudatum

ABSTRACT The reproductive form of a macronucleus-specific symbiont Holospora obtuse , when harbored by the macronucleus of the ciliate Paramecium caudatum , selectively synthesized a 63-kDa protein which is immunologically related to GroEL, or HSP60, of Escherichia coli. Heat shock treatment of isolated cells of the reproductive and infectious form of the bacterium also induced the synthesis of the GroEL homolog. Immunoblotting showed that the amount of this protein per cell, whether the reproductive or infectious form, is roughly constant. Cloning and sequencing of a gene coding for the GroEL homolog suggested that the protein is 55.2% identical to GroEL of E. coli at the amino acid sequence level, and that the gene is preceded by an open reading frame which encodes a protein 39.6% identical to GroES of E. coli. Northern blot hybridization showed that the GroEL homologous gene is highly expressed in the reproductive form, but only in a trace amount in the intermediate and infectious form. Immunoelectron microscopy revealed that the GroEL homolog is localized in the cytoplasm of the reproductive and infectious form.     

Quantitative Changes in Periplasmic Proteins of the Macronucleus-Specific Bacterium Holospora obtusa in the Infection Process of the Ciliate Paramecium caudatum

ABSTRACT. The Gram-negative bacterium Holospora obrusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum. The infectious form of this bacterium infects the host macronucleus through digestive vacuoles and differentiates into the reproductive form two days after the infection in the nucleus. The monoclonal antibodies IF-3–1 and IF-3–2 reacted with 39 and 1S kDa periplasmic proteins, respectively, that were specific for the infectious form of H. obrusa. Because the antigens were not detected in the reproductive form of the bacterium, it appears that expression of the proteins decreases during or soon after the infection. Using these antibodies, quantitative changes in the antigens in the early infection process were examined by immunoblotting and immunogold electron microscopy. Immunoblotting showed that the amounts of both antigens were reduced within 1 h after the bacteria were engulfed into the digestive vacuoles of the paramecia, but that the amounts of IF-3–2 antigens declined earlier than the IF-3–1 antigen. Immunogold labeling showed that the level of IF-3–2 antigens became very low in the bacteria in the host digestive vacuoles, whereas there was no similar decrease in amount of IF-3–1 antigens. Possible functions of the antigens are discussed. The IF-3–1 antigens decrease in concentration in parallel with the decrease in the periplasmic region.     

Developmental expression of macronuclear specific antigen in Paramecium caudatum.

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudatum and P. dubosqui. Immunoblotting showed that the antigen was a polypeptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macronuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus.     

Recovery of the ciliate Paramecium multimicronucleatum following bacterial infection with Holospora obtusa

The macronucleus (Ma) of Paramecium multimicronucleatum can be experimentally infected with bacteria of the species Holospora obtusa, a macronuclear-specific parasite of P. caudatum. However, usually all bacteria disappear from the nucleus within 1–2 days after infection. The results of infecting several different stocks of P. multimicronucleatum with several different isolates of H. obtusa from P. caudatum were studied during some days after infection to investigate this disappearance. Using light, fluorescence and electron microscopy at different stages of bacterial disappearance, it was shown that what we call “cleaning” of the nucleus is a fast and active process. P. multimicronucleatum can be infected with the infectious forms of H. obtusa within 2 h, but the majority of the bacteria were lost from the Ma by 10–19 h. They were released from the host nucleus into the cytoplasm and then to the surrounding medium. At first, the infected Ma shortened and became almost rounded. Before leaving the Ma, the majority of the bacteria somehow assembled into groups and these aggregates, coated with material that appears to be nuclear chromatin, protruded into the cytoplasm and were finally separated from the Ma. Sometimes single bacteria were extruded in the same manner. Bacterial release from infected Ma can be stopped by low temperature and is delayed and reduced by nocodazole treatment suggesting that intra-macronuclear microtubules may be involved.     

Analysis of natural diversity of symbiotic relationships in the Paramecium bursaria--Holospora curviuscula system

Bacteria of the genus Holospora belong to obligatory endonucleobionts of ciliates of the genus Paramecium. The bacteria show specificity towards the particular host species and the types of nuclei they infect: macro- or micronuclei. During a long-term screening of P. bursaria clones, belonging to three different syngens, Holospora inhibited cells of two syngens only. Using the number of host clones and symbiont isolates, it was shown that H. curviuscula was unable to pass successfully through the syngen barrier even under experimental infection. Considering the species level of specificity in Holospora associations of P. caudatum we suggest the existence of a greater evolutionary divergence in P. bursaria syngens than in syngens of P. caudatum. We have revealed that in incompatible combinations "host clone--symbionts isolate" the complicated bacterial life cycle may be blocked at definite stages depending on genetic features of both partners. Thus, the recognition of the full block spectrum could break the continuous infection process down to independently controlled steps. The block spectrum revealed in the system of P. bursaria--H. curviuscula demonstrates its significant similarity to block spectra of other systems within the Holospora--Paramecium complex. A block of transverse binding formation has been first revealed in Holospora dividing in the nucleus.     

Developmentally Controlled Rearrangement of Surface Protein Genes in Paramecium tetraurelia1

ABSTRACT Early research on Paramecium genetics highlighted the role of the cytoplasm on inheritance. Today this tradition continues as recent investigations of macronuclear development in Paramecium have revealed unusual cytoplasmic effects that are not easily explained within current paradigms. It is generally assumed that most programmed DNA rearrangements in ciliates are regulated by cis acting signals encoded within the germline (micronuclear) DNA, but there are increasing examples in which the old macronucleus acts through the cytoplasm (in trans) to affect the loss and rearrangement of DNA in the developing macronucleus. The remarkable specificity of this effect has forced a reevaluation of the standard view of macronuclear determination in Paramecium. This review summarizes our knowledge of the effect of the old macronucleus on the developmentally controlled rearrangements of the P. tetraurelia, stock 51A and B variable surface protein genes.     

Endosymbionts in paramecium

Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holospora's invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium.     

The route of a bacterium Holospora in the cell of Paramecium (Ciliophora,Protista) from phagosome to the nucleus

Problems encountered at the initial stages of stable symbiotic system formation are discussed in the review. The most studied models for interaction between pathogenic bacteria and metazoan cells are compared with a similar system including Paramecium (a ciliatte)--Holospora (a bacterium). Literary and our own data on the infection of P. caudatum with specific endocytobionts inhabiting the nuclear apparatus (H. obtusa in the macronucleus), and H. undulata (in the micronucleus) are analysed with respect to the modern understanding of the intracellular vesicle trafficking.     

Electron microscope study of the structure of Holospora caryophila

The ultrastructure of the symbiont Holospora caryophila found in the macronucleus of Paramecium biaurelia has been studied in the electron microscope following preparation by thin sections, negative staining and shadow-casting techniques. Holospora were identified by their characteristic morphology of slender elongated cells when seen in whole mounts in negative stain or contrasted with shadowing. The long forms were seen to possess a helical configuration, also an asymmetrical shape.Thin sections of Holospora revealed the cell envelope structure consistent with a Gram-negative organism. A complex network of internal membranes, together with a relatively electron transparent region at one end of the cytoplasm, was frequently observed.