Cytokinin biosynthesis in crown gall tumors

Using combined HPLC-RIA, cytokinin levels in a series of tobacco crown gall tumors, having altered morphologies as a result of transposon mutagenesis were examined. Average trans-ribosylzeatin levels in the shoot-producing tumors (tms mutants) and root-producing tumors (tmr mutants) were 1400 pmol/gm and 0.97 pmol/gm, respectively. Cytokinin/auxin ratios were highest in the tms mutants (17-24) and lowest in the tmr mutants (0.004). The evidence suggests that the T-DNA loci which define tumor morphology, also determine phytohormone levels. Cytokinin levels were also examined in several octopine crown gall tumors, with different T[subscript L] and T[subscript R] DNA copy numbers. The trans-ribosylzeatin level in the high T[subscript L] DNA copy number tumor line, 15955/01, was 437 ng/gm compared to 6-30 ng/gm in the other low T[subscript L] DNA copy number tumor lines, suggesting that T-DNA copy number can influence cytokinin levels. At a detection limit of 100 pg/gm, no trans-ribosylzeatin was detected in untransformed tobacco tissue. Some of the properties of the cytokinin biosynthetic enzyme, dimethylallylpyrophosphate:adenosine 5'-monophosphate dimethylallyl transferase (DMA transferase), from Nicotiana tabacum crown gall tumor line 15955/01 were studied. This enzyme demonstrated a substrate specificity for adenosine 5'-monophosphate and catalyzed the synthesis of iso-pentenyladenosine 5'-monophosphate (iPA-P). DMA transferase activity from cell-free extracts of four crown gall tumors and untransformed tobacco tissue was estimated by an assay which included high performance liquid chromatography and immunoaffinity chromatography. No enzyme activity was detected in untransformed tobacco tissue, but was present in all four crown gall tumor lines. Specific activities (1 Unit is defined as the synthesis of 1 fmol iPA-P/min/mg total protein) of the enzyme in the crown gall tumor lines were: A6S/2, 2.0; B₆ 806/E9, 6.6; 15955/1, 2.7 and 15955/01, 5.7. The results strongly indicate that the T-DNA regulates non-tRNA mediated biosynthesis of cytokinins

The reduced genome of the parasitic microsporidian Enterocytozoon bieneusi lacks genes for core carbon metabolism

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial 2.5 License. The definitive version was published in Genome Biology and Evolution 2 (2010): 304, doi:10.1093/gbe/evq022.Reduction of various biological processes is a hallmark of the parasitic lifestyle. Generally, the more intimate the association between parasites and hosts the stronger the parasite relies on its host's physiology for survival and reproduction. However, some systems have been held to be indispensable, for example, the core pathways of carbon metabolism that produce energy from sugars. Even the most hardened anaerobes that lack oxidative phosphorylation and the tricarboxylic acid cycle have retained glycolysis and some downstream means to generate ATP. Here we describe the deep-coverage genome resequencing of the pathogenic microsporidiian, Enterocytozoon bieneusi, which shows that this parasite has crossed this line and abandoned complete pathways for the most basic carbon metabolism. Comparing two genome sequence surveys of E. bieneusi to genomic data from four other microsporidia reveals a normal complement of 353 genes representing 30 functional pathways in E. bieneusi, except that only 2 out of 21 genes collectively involved in glycolysis, pentose phosphate, and trehalose metabolism are present. Similarly, no genes encoding proteins involved in the processing of spliceosomal introns were found. Altogether, E. bieneusi appears to have no fully functional pathway to generate ATP from glucose. Therefore, this intracellular parasite relies on transporters to import ATP from its host.This work was supported by grants from the Canadian Institutes for Health Research (MOP-84265), the National Institutes of Health (NIH AI31788, R21 AI52792, and R21 AI064118), and the National Science Foundation (MCB- 0135272). N.C. is a Scholar of the Canadian Institute for Advanced Research and is supported by a fellowship from the Swiss National Science Foundation (NSF) (PA00P3- 124166). D.E. is supported by the Swiss NSF. P.J.K. is a Fellow of the Canadian Institute for Advanced Research and a Senior Scholar of the Michael Smith Foundation for Health Research

Genomic survey of the non-cultivatable opportunistic human pathogen, Enterocytozoon bieneusi

© 2009 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS Pathogens 5 (2009): e1000261, doi:10.1371/journal.ppat.1000261.Enterocytozoon bieneusi is the most common microsporidian associated with human disease, particularly in the immunocompromised population. In the setting of HIV infection, it is associated with diarrhea and wasting syndrome. Like all microsporidia, E. bieneusi is an obligate, intracellular parasite, but unlike others, it is in direct contact with the host cell cytoplasm. Studies of E. bieneusi have been greatly limited due to the absence of genomic data and lack of a robust cultivation system. Here, we present the first large-scale genomic dataset for E. bieneusi. Approximately 3.86 Mb of unique sequence was generated by paired end Sanger sequencing, representing about 64% of the estimated 6 Mb genome. A total of 3,804 genes were identified in E. bieneusi, of which 1,702 encode proteins with assigned functions. Of these, 653 are homologs of Encephalitozoon cuniculi proteins. Only one E. bieneusi protein with assigned function had no E. cuniculi homolog. The shared proteins were, in general, evenly distributed among the functional categories, with the exception of a dearth of genes encoding proteins associated with pathways for fatty acid and core carbon metabolism. Short intergenic regions, high gene density, and shortened protein-coding sequences were observed in the E. bieneusi genome, all traits consistent with genomic compaction. Our findings suggest that E. bieneusi is a likely model for extreme genome reduction and host dependence.This research was supported by National Institutes of Health (NIH) grants R21 AI064118 (DEA) and R21 AI52792 (ST). HGM was supported in part by NIH contracts HHSN266200400041C and HHSN2662004037C (Bioinformatics Resource Centers) and by the G. Unger Vetlesen Foundation

Patterns of Genome Evolution among the Microsporidian Parasites Encephalitozoon cuniculi, Antonospora locustae and Enterocytozoon bieneusi

Microsporidia are intracellular parasites that are highly-derived relatives of fungi. They have compacted genomes and, despite a high rate of sequence evolution, distantly related species can share high levels of gene order conservation. To date, only two species have been analysed in detail, and data from one of these largely consists of short genomic fragments. It is therefore difficult to determine how conservation has been maintained through microsporidian evolution, and impossible to identify whether certain regions are more prone to genomic stasis.Here, we analyse three large fragments of the Enterocytozoon bieneusi genome (in total 429 kbp), a species of medical significance. A total of 296 ORFs were identified, annotated and their context compared with Encephalitozoon cuniculi and Antonospora locustae. Overall, a high degree of conservation was found between all three species, and interestingly the level of conservation was similar in all three pairwise comparisons, despite the fact that A. locustae is more distantly related to E. cuniculi and E. bieneusi than either are to each other.Any two genes that are found together in any pair of genomes are more likely to be conserved in the third genome as well, suggesting that a core of genes tends to be conserved across the entire group. The mechanisms of rearrangments identified among microsporidian genomes were consistent with a very slow evolution of their architecture, as opposed to the very rapid sequence evolution reported for these parasites

Rapid Displacement of Cryptosporidium parvum Type 1 by Type 2 in Mixed Infections in Piglets

Genotypes 1 and 2 of Cryptosporidium parvum are the primary types associated with infections in humans, with type 1 being by far the predominant genotype. The frequency of mixed infection with both genotypes in humans is relatively rare, while type 1, which experimentally infects other mammals, has been found to naturally infect almost exclusively humans. One possible explanation for the absence of type 1 in other mammals and the low frequency of mixed infections in humans is the inability of type 1 to compete with type 2 in nature when both occur simultaneously. To investigate this, we challenged gnotobiotic piglets with equal number of oocysts of type 1 and type 2, given either simultaneously or with type 2 given 24 or 48 h after type 1. The genotype of the oocysts excreted in feces and the relative distribution of each of the genotypes throughout the gut at necropsy were determined. Regardless of the time interval between challenges with the two genotypes, type 2 invariably displaced type 1. The rate of displacement was rapid when both genotypes were given simultaneously, after which no traces of type 1 were detected in the feces or in gut sections by PCR. Infection with type 1 24 or 48 h before challenge with type 2, while permitting type 1 to become established, was still rapidly eliminated within 3 days after challenge with type 2. These observations have major implications regarding the relative perpetuation and survival of these two genotypes in mammals

Characterization of Cryptosporidium meleagridis of Human Origin Passaged through Different Host Species

Cryptosporidium meleagridis, a protozoon first observed in turkeys, has been linked by several investigators to cryptosporidiosis in humans. C. meleagridis is the only known Cryptosporidium species that infects both avian and mammalian species. We describe the successful propagation of C. meleagridis (isolate TU1867), originally purified from a patient with diarrhea, in laboratory animals including chickens, mice, piglets, and calves. TU1867 was readily transmitted from one animal host to another, maintaining genetic homogeneity and stability. The rate of infectivity and virulence of TU1867 for the mammalian species were similar to those of Cryptosporidium parvum. Laboratory propagation of genetically and phenotypically stable and well-characterized reference isolates, representing various Cryptosporidium species, particularly those infectious to humans, will improve considerably the spectrum and quality of laboratory and field investigations on this medically important protozoa