Circular Ribosomal RNA Genes Are a General Feature of Schizopyrenid Amoebae

ABSTRACT We have recently shown that the ribosomal RNA genes of the amoebo-flagellate Naegleria gruberi Schardinger, 1899, strain NEG-M are carried exclusively on a 14 kilobasepair plasmid. To explore the distribution of this unique gene arrangement, we have examined another strain of N. gruberi and four other species from the order Schizopyrenida. All have this unusual gene arrangement although the size of the plasmid varies widely. Species groups based on morphological criteria do not agree with those resulting from comparison of plasmid restriction enzyme patterns.     

Large genetic heterogeneity within amoebas of the species Naegleria gruberi and evolutionary affinities to the other species of the genus

The allozyme survey was extended to 7 strains of Naegleria gruberi and N. jadini in order to further characterize the genetic structure of these free-living amoebas. As formerly known for several characters the electrophoretic evidence reveals considerable heterogeneity at the genetic level among N. gruberi strains. Moreover, 2 distinct gene pools, that might likely represent natural taxa, are clearly identified. The single strain of N. jadini appears evolutionarily related to 1 group of N. gruberi which is also related to N. a. australiensis.     

Restriction endonuclease analysis of mitochondrial DNA as an aid in the taxonomy of Naegleria and Vahlkampfia

Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.     

Large Genetic Heterogeneity Within Amoebas of the Species Naegleria gruberi and Evolutionary Affinities to the Other Species of the Genus

The allozyme survey was extended to 7 strains of Naegleria gruberi and N. jadini in order to further characterize the genetic structure of these free-living amoebas. As formerly known for several characters the electrophoretic evidence reveals considerable heterogeneity at the genetic level among N. gruberi strains. Moreover, 2 distinct gene pools, that might likely represent natural taxa, are clearly identified. The single strain of N. jadini appears evolutionarily related to 1 group of N. gruberi which is also related to N. a. australiensis.     

Geographic origin and spread of pathogenic Naegleria fowleri deduced from restriction enzyme patterns of repeated DNA

The restriction enzyme patterns of repeated DNA from 20 Naegleria fowleri and 13 N. gruberi strains were compared. On this basis strains of N. fowleri could be easily separated from N. gruberi. Although the restriction enzyme profiles of N. fowleri strains are quite homogenous, strains originating from Europe and from Australia had slightly different patterns. Both European and Australian profiles were found in the USA. In Australia a genetic variant was detected that is the same as the N. fowleri type present in New Zealand. Profiles of strains from India were identical with those from Europe. These results give additional information on the probable origin and dispersal of N. fowleri in the world. Strains of N. gruberi exhibit much more heterogenous banding patterns. Four European N. gruberi strains were, however, nearly identical while two strains from New Zealand had only a single band difference with one restriction enzyme. One Australian strain of N. gruberi was identical to an American strain that has been in culture for almost 30 years. The presence of a virus-like particle in a sister strain of this American isolate did not affect its banding patterns. Other strains of N. gruberi from the USA had diverse restriction enzyme patterns. Adelphamoeba galeacystis has restriction enzyme profiles distinct from those of the Naegleria strains investigated. Isoenzyme analysis in agarose isoelectric focusing confirmed the existence of intraspecific differences in N. fowleri.     

Lactococcal plasmid pWV01 as an integration vector for lactococci.

A Bacillus subtilis strain was constructed that contained the repA gene of the lactococcal plasmid pWVO1 in its chromosome. This strain was used to construct the pWVO1-based integration vector pINT1, which lacked the repA gene. The 3.6-kb plasmid pINT1 was not able to replicate in Lactococcus lactis MG1363 but integrated into the chromosome via a Campbell-like mechanism when a lactococcal chromosomal DNA fragment was incorporated in the plasmid. Transformants were obtained that carried between one and four plasmid copies, in stable tandem arrangement on the chromosome. The results indicate that pWVO1 can be used for the development of a Campbell-like integration system fully derived of lactococcal DNA, with which stable multiple copies of any gene of interest can be generated in the lactococcal chromosome.     

Lactococcal plasmid pWV01 as an integration vector for lactococci.

A Bacillus subtilis strain was constructed that contained the repA gene of the lactococcal plasmid pWVO1 in its chromosome. This strain was used to construct the pWVO1-based integration vector pINT1, which lacked the repA gene. The 3.6-kb plasmid pINT1 was not able to replicate in Lactococcus lactis MG1363 but integrated into the chromosome via a Campbell-like mechanism when a lactococcal chromosomal DNA fragment was incorporated in the plasmid. Transformants were obtained that carried between one and four plasmid copies, in stable tandem arrangement on the chromosome. The results indicate that pWVO1 can be used for the development of a Campbell-like integration system fully derived of lactococcal DNA, with which stable multiple copies of any gene of interest can be generated in the lactococcal chromosome.     

rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid.

An extrachromosomal DNA was discovered in Naegleria gruberi. The 3,000 to 5,000 copies per cell of this 14-kilobase-pair circular plasmid carry all the 18S, 28S, and 5.8S rRNA genes. The presence of the ribosomal DNA of an organism exclusively on a circular extrachromosomal element is without precedent, and Naegleria is only the third eucaryotic genus in which a nuclear plasmid DNA has been found.     

Euglena gracilis chloroplast transfer RNA transcription units. Nucleotide sequence analysis of a tRNAThr-tRNAGly-tRNAMet-tRNASer-tRNAGln gene cluster

The arrangement and the nucleotide sequence of the tRNA genes in the 2.0-kilobase-pair EcoRI restriction fragment EcoQ of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. This fragment, cloned in pBR325 to form the plasmid pEZC300, contains five tRNA genes. The DNA insert of this plasmid, a known tRNA gene locus (Orozco, E.M., Jr., and Hallick, R.B. (1982) J. Biol. Chem. 257, 3258-3264) has been mapped by Southern gel analysis using a 32P-labeled oligodeoxynucleotide tRNA gene probe. The DNA sequence of 870 base pairs (bp) from EcoQ containing the entire tRNA gene locus was determined. The organization of this tRNA gene cluster on the E. gracilis chloroplast chromosome is tRNAUUGGln-14-BP spacer-RNAGCUSer-175-bp spacer-tRNACAUMet-12-bp spacer-tRNAGCCGly-5-bp spacer-tRNAUGUThr. The tRNAUUGGln and tRNAGCUSer gene sequences are of the opposite polarity as the other three gene sequences, but of the same polarity as the rRNA genes. The tRNAMet gene is a putative initiator tRNA. The five tRNA genes are separated and flanked by A-T-rich spacer sequences. This gene arrangement is consistent with the model that E. gracilis chloroplast tRNA genes are transcribed into multicistronic tRNA precursors. The DNA sequences have been used to deduce the primary and secondary structures of the tRNAs.     

Genetic evidence for a dystrophin-glycoprotein complex (DGC) in Caenorhabditis elegans

A novel alpha-tubulin gene (alpha6) was cloned from a genomic library of Naegleria gruberi strain NB-1 and characterized. The open reading frame of alpha6 contained 1359 nucleotides encoding a protein of 452 amino acids (aa) with a calculated molecular weight of 50.5 kDa. The nucleotide sequence of the open reading frame of alpha6 showed considerable divergence (68.4% identity) when compared with previously cloned N. gruberi alpha-tubulin genes, which share about 97% identity in DNA sequences. The deduced aa sequence of alpha6-tubulin was 61.9% identical to that of alpha13-tubulin, which was cloned from the same strain, and showed similar identities to those of alpha-tubulins from other species (54 approximately 62%). These data showed that alpha6-tubulin is one of the most divergent alpha-tubulins so far known. Alpha6-tubulin was found to be expressed in actively growing cells and repressed quickly when these cells were induced to differentiate. Immunostaining with an antibody against alpha6-tubulin showed that alpha6-tubulin is present in the nuclei and mitotic spindle-fibers but absent in flagellar axonemes or cytoskeletal microtubules. These data finally established the presence of an alpha-tubulin that is specifically utilized for spindle-fiber microtubules and distinct from the flagellar axonemal alpha-tubulins in N. gruberi, hence confirmed the multi-tubulin hypothesis in this organism.     

Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification

To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.     

Electrophoretic karyotype and linkage groups of the amoeboflagellate Naegleria gruberi

We have constructed a molecular karyotype for two strains of Naegleria gruberi using pulsed field gel electrophoresis. Each strain has about 23 chromosomes, considerably more than any previous estimate. These chromosomes range in size from 400 kilobasepairs to over 2,000 kilobasepairs. In Naegleria, construction of the DNA karyotype depends on assessment of the anomalous electrophoretic mobility of the circular ribosomal RNA genes. We have determined the chromosomal locations of an identified unique gene (flagellar calmodulin) and four identified multigene families (alpha- and beta-tubulin, actin, ubiquitin), as well as three differentially expressed genes of unknown functions. The ca. 12 actin genes are dispersed over at least seven chromosomes, whereas the majority of the more than eight alpha-tubulin genes are confined to a single chromosome. The ubiquitin genes are found on five chromosomes in one strain and seven in the other and the beta-tubulin genes are on three or four. Our observations provide a foundation for molecular genetic studies in this organism.     

Electrophoretic Karyotype and Linkage Groups of the Amoeboflagellate Naegleria gruberi

ABSTRACT. We have constructed a molecular karyotype for two strains of Naegleria gruberi using pulsed field gel electrophoresis. Each strain has about 23 chromosomes, considerably more than any previous estimate. These chromosomes range in size from 400 kilobasepairs to over 2,000 kilobasepairs. In Naegleria , construction of the DNA karyotype depends on assessment of the anomalous electrophoretic mobility of the circular ribosomal RNA genes. We have determined the chromosomal locations of an identified unique gene (flagellar calmodulin) and four identified multigene families (α- and β-tubulin, actin, ubiquitin), as well as three differentially expressed genes of unknown functions. The ca. 12 actin genes are dispersed over at least seven chromosomes, whereas the majority of the more than eight α-tubulin genes are confined to a single chromosome. The ubiquitin genes are found on five chromosomes in one strain and seven in the other and the β-tubulin genes are on three or four. Our observations provide a foundation for molecular genetic studies in this organism.     

Small-subunit ribosomal RNA sequence from Naegleria gruberi supports the polyphyletic origin of amoebas

We have sequenced the small-subunit ribosomal RNA gene of the amoebo- flagellate protozoan Naegleria gruberi. Comparison of this sequence with the rRNA sequences of other eukaryotes resulted in a phylogenetic tree that supports the suggested polyphyletic origin of amoebas and suggests a flagellate ancestry for Naegleria.      

Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri

Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica-clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri.     

Intranasal immunization of mice against Naegleria fowleri

The purpose of this research was to determine whether mice could be protected from lethal challenge with Naegleria fowleri by prior intranasal exposure to pathogenic and nonpathogenic Naegleria. Mortality ranged from 0 to 100% for mice inoculated intranasally (i.n.) with 5 x 10(3) amebae of 13 human isolates of N. fowleri. Mice were immunized and challenged i.n. using live amebae of strains of low, medium, and high virulence. The greatest protection against lethal challenge was afforded by three immunizing doses of 10(3) amebae per dose of the strain of medium virulence. Nonpathogenic N. gruberi also was used to immunize mice i.n. against lethal challenge with N. fowleri. Protection was greater following immunization with N. gruberi than it was after immunization with N. fowleri, suggesting that nonpathogenic N. gruberi may be a better immunogen in protecting mice against lethal naeglerial challenge.