Histone-antihistone interactions in the <Emphasis Type="Italic">Escherichia coli-Tetrahymena pyriformis</Emphasis> association

Using the Escherichia coli-Tetrahymena pyriformis model system, we revealed the involvement of bacterial antihistone activity and protozoan histones in interactions between pro-and eukaryotic microorganisms. Antihistone activity enhanced the viability of E. coli in association with T. pyriformis, according to our data on the dynamics of E. coli cell numbers. The strain with antihistone activity induced incomplete phagocytosis in the infusorians, resulting in cytological changes and ultrastructural alterations that indicated the retention of bacterial cells in phagosomes. Bacteria with antihistone activity located in the T. pyriformis cytoplasm influenced the eukaryotic nucleus. This was accompanied by in macronucleus decompactization and a decrease in the average histone content in the population of infusorians. The data obtained suggest that protozoan histone inactivation by bacteria is one of the mechanisms involved in prokaryote persistence in associations with eukaryotic microorganisms.     

Continuous culture of the ciliate Tetrahymena pyriformis on Escherichia coli

The ciliated protozoan Tetrahymena pyriformis was grown in a chemostat fed with a culture of Escherichia coli overflowing from another chemostat. Densities of the protozoan and bacterial populations, mean volume of protozoan cells, yields of protozoan volumes and numbers, and filtering rates of protozoans per cell and per unit volume of biomaterial were determined at five different dilution rates. The data obtained supplement other data already available for the popular test organism T. pyriformis, and they are also comparable with data available for related ciliates.     

Effect of natural amino acids and sugars on cyclase activities in infusoria Tetrahymena pyriformis and Dileptus anser

Natural amino acids and sugars in intracellular eukaryotes are known to regulate adenylyl cyclase (AC) and guanylyl cyclase (GC) systems that control the most important cell processes. The goal of the present work consisted in study of effects of natural amino acids and sugars and some of their derivatives on AC and GC activities of infusoria Tetrahymena pyriformis and Dileptus anser. Methionine, arginine, lysine, and tryptamine stimulated basic AC activity of T. pyriformis, whereas alanine, thyrosine, and cysteine decreased it. Methionine, glycine, alanine, thyrosine, arginine, and to the lesser degree tryptamine and histidine stimulated AC of D. anser. The GC activity of T. pyriformis are increased in the presence of tryptamine, tryptophane, histidine, arginine, and lysine, whereas glycine and aspartic acid, on the contrary, decreased it. Tryptamine, tryptophan, leucine, glutamic acid, serine, histidine, and alanine stimulated the GC activity of D. anser. Glucose, fructose, and sucrose stimulated the basal AC activity of both infusorians and GC of T. pyriformis, with glucose and sucrose increasing AC of T. pyriformis twice, while that of D. anser 4.5 times. Lactose stimulated AC and GC of T. pyriformis and was inefficient with respect to the D. anser cyclases, whereas mannose and galactose did not affect the enzyme activities in both infusorians. The study of the chemotactic response of infusorians to amino acids and sugars indicates that involved in realization of this response can be signaling pathways both dependent on and independent of cyclic nucleotides. Thus, it has been established for the first time that several amino acids and sugars affect functional activity of enzymes with cyclase activity of the infusorians T. pyriformis and D. anser. This confirms the hypothesis that at early stages of evolution the large spectrum of comparatively simple natural molecules has a hormone-like action.     

Characteristics of the interaction of legionellae and Tetrahymena pyriformis

L. pneumophila avirulent strains have been shown to lose their capacity for multiplication in T. pyriformis, while the concentration of the virulent strain increases 1000-fold. The loss of the hemolytic activity of L. pneumophila virulent strain leads to the loss of its capacity for multiplication in infusorians.     

Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.     

Effect of Protozoa on Bacterial Degradation of an Aromatic Compound

Prototrophic and growth factor-requiring strains of Alcaligenes spp. were used to study the effect of a protozoan, Tetrahymena pyriformis, on the degradation of p-aminobenzoate. The protozoan inhibited activity of the prototrophic bacterium by reducing its population size. For the growth factor-requiring strain of Alcaligenes, T. pyriformis provided the required growth factors so that the predator permitted the bacteria to grow and to continue p-aminobenzoate degradation. T. pyriformis inhibited bacterial activity when the amino acid supply was in excess, but activity of the auxotrophic strain of Alcaligenes was stimulated by the protozoan when the amino acid supply was limiting, although the bacterial population size was reduced by the protozoan.     

Interactions of pseudomonas aeruginosa hemagglutinins with Euglena gracilis, Chlamydomonas reinhardi, and Tetrahymena pyriformis

The galactosephilic and mannosephilic hemagglutinins of Pseudomonas aeruginosa adsorbed onto Euglena gracilis, Chlamydomonas reinhardi, and Tetrahymena pyriformis. Furthermore, peroxidase binding to the 3 protozoan species was shown to be mediated by these lectins. Binding of Pseudomonas lectins to E. gracilis and C. reinhardi caused their specific agglutination, whereas no agglutination was observed with T. pyriformis, even after treatment by papain or by NaF. Added to the culture medium, the Pseudomonas hemagglutins stimulated growth of E. gracilis and T. pyriformis due to their binding to these protozoa; this effect was partly inhibited by the specific sugar.     

Interactions of Pseudomonas Aeruginosa Hemagglutinins With Euglena Gracilis, Chlamydomonas Reinhardi, and Tetrahymena Pyriformis

SYNOPSIS The galactosephilic and mannosephilic hemagglutinins of Pseudomonas aeruginosa adsorbed onto Euglena gracilis, Chlamydomonas reinhardi , and Tetrahymena pyriformis . Furthermore, peroxidase binding to the 3 protozoan species was shown to be mediated by these lectins. Binding of Pseudomonas lectins to E. gracilis and C. reinhardi caused their specific agglutination, whereas no agglutination was observed with T. pyriformis , even after treatment by papain or by NaF. Added to the culture medium, the Pseudomonas hemagglutinins stimulated growth of E. gracilis and T. pyriformis due to their binding to these protozoa: this effect was partly inhibited by the specific sugar.     

Protozoan feeding and bacterial wall growth.

Monod's model is often assumed to describe the kinetics of feeding of a protozoan population on a bacterial population in a chemostat. An earlier study (J. L. Jost et al., J. Bacteriol., 113, 84 (1973)) of the feeding of Tetrahymena pyriformis on either Escherichia coli or Azotobacter vinelandii found that this model correctly predicted the occurrence of sustained oscillations of population densities but made predictions of minimum bacterial population densities that were much smaller than those observed. The earlier study removed the discrepancy between the model and data by replacing Monod's model with a different model. It is shown in the present study that the discrepancy can be explained equally as well if Monod's model for the feed relation is retained and if, in addition, growth of bacteria on the chemostat walls is allowed for in the model equations.     

Survival of coliforms and bacterial pathogens within protozoa during chlorination.

The susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria. Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were greater than 50-fold more resistant to free chlorine when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for survival of fastidious bacteria in dilute and inhospitable aquatic environments.     

Subcellular distribution of calmodulin and calmodulin-binding sites in Tetrahymena pyriformis

The subcellular distribution of calmodulin and particulate calmodulin-binding activity was studied in a eukaryotic protozoan, Tetrahymena pyriformis NT-1. The particulate calmodulin-binding activity was found to be localized principally in microsomes and to some extent in cilia and surface membranes called pellicles. Nearly all (93%) of the total amount of calmodulin was recovered in two soluble compartments, the ciliary and postmicrosomal supernatant fractions.     

High mobility group nonhistone chromosomal proteins also exist in Tetrahymena.

High mobility group (HMG) nonhistone chromosomal proteins have been shown to exist also in the ciliated protozoan Tetrahymena pyriformis. One or two histone-like components were extracted with 0.25 M HCl from the chromatin, in addition to five histone species. These proteins were also extracted selectively with 0.5 M HClO4, 0.35 M NaCl, or 4 mM spermidine, together with H1 histone, and were characterized as HMG proteins on the basis of the following criteria: high mobilities on polyacrylamide gel electrophoresis, relatively low molecular weights, amino acid compositions rich in lysine and glutamic acid, and relative contents in chromatin. This extends the distribution of the HMG proteins to all four eukaryotic kingdoms, and suggests the possibility that they have some universal role in chromatin structure and function.     

The histone content of Tetrahymena ribosomal gene-containing chromatin

The histone composition of the ribosomal gene containing chromatin (rChromatin) of the ciliated protozoan Tetrahymena pyriformis has been investigated using purified nucleolar fractions in which ribosomal DNA constitutes 65-82% of the total DNA. In isolated nucleoli, rChromatin largely retains the periodic activated structure characteristic of rChromatin in the nucleus. For all five major histone classes, the histone to DNA ratios of nucleolar chromatin are similar to those of bulk macronuclear chromatin. These results argue that the differences between activated rChromatin and inactivated chromatin are not due to a deficiency in the number of histones available to form nucleosomes on the rDNA.     

Molecular cloning and expression of hctB encoding a strain-variant chlamydial histone-like protein with DNA-binding activity.

Two DNA-binding proteins with similarity to eukaryotic histone H1 have been described in Chlamydia trachomatis. In addition to the 18-kDa histone H1 homolog Hc1, elementary bodies of C. trachomatis possess an antigenically related histone H1 homolog, which we have termed Hc2, that varies in apparent molecular mass among strains. We report the molecular cloning, expression, and nucleotide sequence of the hctB gene encoding Hc2 and present evidence for in vivo DNA-binding activity of the expressed product. Expression of Hc2 in Escherichia coli induces a compaction of bacterial chromatin that is distinct from that observed upon Hc1 expression. Moreover, isolated nucleoids from Hc2-expressing E. coli exhibit markedly reduced sensitivity to DNase I. These properties of Hc2 are consistent with a postulated role in establishing the nucleoid structure of elementary bodies.     

Functional similarities and differences of an archaeal Hsp70(DnaK) stress protein compared with its homologue from the bacterium Escherichia coli

Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes. Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception. Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues. The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function. The M. mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2. Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M. mazei DnaK. The M. mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison. However, in vivo analyses indicate that there are also significant differences. The M. mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M. mazei dnaK gene. Thus, while the data from in vitro tests demonstrate functional similarities between the M. mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ.     

UV inactivation of pathogenic and indicator microorganisms

Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts.     

UV inactivation of pathogenic and indicator microorganisms.

Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts.     

Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains.

Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli. We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues. This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E. coli cells for subsequent infection and propagation. This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E. coli locus responsible for this difference in efficiency. It was determined that the E. coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrB1 mutation, which inactivates the classic McrB restriction activity. (This mutation was verified by sequence analysis.) However, this McrB1 activity is not observed when the cloned mcrB1 gene is inserted into the E. coli genome at one copy per chromosome. A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome. The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences. Removal of this DNA region including the mrr gene from E. coli K-12 strains allows high rescue efficiencies equal to those of E. coli C strains. These modified E. coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.     

Allosteric activation by purine nucleosides and nucleotides of the inactive by phosphatase ornithine decarboxylase of Tetrahymena pyriformis

Ornithine decarboxylase (ODC) of Tetrahymena pyriformis is inactivated by E. coli or calf intestinal alkaline phosphatase. Inactivated ODC by E. coli alkaline phosphatase, form b, can be allosterically reactivated to form a by purine-nucleosides or -nucleotides at 10(-4) M concentration. Inactivated ODC by calf intestinal alkaline phosphatase bound to agarose can be converted to form a by purine-analogues with 10(-7) M concentration only if inorganic phosphate is included in the incubation mixture. Pyridoxal phosphate, phosphoamino acids or other phospho-compounds are incapable to promote the conversion of b form to a form of ODC. These data suggest that purine-analogues may be the in vivo physiological regulators of ODC of T. pyriformis.