Alejandro D. Nusblat

Detection and characterization of quorum sensing signal molecules in Acinetobacter strains

Quorum sensing is a widespread regulatory mechanism among Gram-negative bacteria. In this study, Acinetobacter strains were assayed for the presence of quorum sensing signal molecules capable of activating N-acylhomoserine lactone biosensors. By using an Agrobacterium tumefaciens reporter strain it was shown that all the cultures produced two to four detectable signal molecules with different chromatographic patterns. In A. calcoaceticus BD413 supernatants four compounds were detected in a time-dependent manner, and maximal activity was reached at stationary phase. The number of signal molecules was dependent on medium composition; typically, cultures in minimal medium displayed one or two more signals, as compared to complex medium. None of the Acinetobacter supematants showed autoinduction activity with an Chromobacterium violaceum reporter strain, neither in direct or competition assays.

Characterization and Properties of Cholesterol Desaturases from the Ciliate Tetrahymena thermophila

Abstract. Live Tetrahymena thermophila transforms exogenous cholesterol into 7,22‐bis, dehydrocholesterol (DHC) by desaturation at positions C7(8) and C22(23) of the cholesterol moiety. In this first report on expression, isolation, characterization, and reconstitution of Tetrahymenas cholesterol desaturases in cell‐free extracts, we describe conditions for increasing the expression of both desaturases based on the addition of specific sterols to the culture medium. Reactions performed in vitro, with isolated microsomes, yield only the mono‐unsaturated derivatives, 7‐DHC and/or 22‐DHC. However, selectivity towards one product can be improved with the addition of specific compounds: β‐mercaptoethanol inhibited C22(23) desaturase activity completely, while ethanol selectively increased this activity. Detergent‐solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroyl‐phosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b5. NADH or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions.

Conservation and innovation in Tetrahymena membrane traffic: proteins, lipids, and compartments

The past decade has seen a significant expansion in our understanding of membrane traffic in Tetrahymena thermophila, facilitated by the development of new experimental tools and by the availability of the macronuclear genome sequence. Here we review studies on multiple pathways of uptake and secretion, as well as work on metabolism of membrane lipids. We discuss evidence for conservation versus innovation in the mechanisms used in ciliates compared with those in other eukaryotic lineages, and raise the possibility that existing gene expression databases can be exploited to analyze specific pathways of membrane traffic in these cells.

The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b(5), Cyt b(5) reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes.

A Novel Sterol Desaturase-Like Protein Promoting Dealkylation of Phytosterols in Tetrahymena thermophila

ABSTRACT The gene TTHERM_00438800 (DES24) from the ciliate Tetrahymena thermophila encodes a protein with three conserved histidine clusters, typical of the fatty acid hydroxylase superfamily. Despite its high similarity to sterol desaturase-like enzymes, the phylogenetic analysis groups Des24p in a separate cluster more related to bacterial than to eukaryotic proteins, suggesting a possible horizontal gene transfer event. A somatic knockout of DES24 revealed that the gene encodes a protein, Des24p, which is involved in the dealkylation of phytosterols. Knocked-out mutants were unable to eliminate the C-24 ethyl group from C29 sterols, whereas the ability to introduce other modifications, such as desaturations at positions C-5(6), C-7(8), and C-22(23), were not altered. Although C-24 dealkylations have been described in other organisms, such as insects, neither the enzymes nor the corresponding genes have been identified to date. Therefore, this is the first identification of a gene involved in sterol dealkylation. Moreover, the knockout mutant and wild-type strain differed significantly in growth and morphology only when cultivated with C29 sterols; under this culture condition, a change from the typical pear-like shape to a round shape and an alteration in the regulation of tetrahymanol biosynthesis were observed. Sterol analysis upon culture with various substrates and inhibitors indicate that the removal of the C-24 ethyl group in Tetrahymena may proceed by a mechanism different from the one currently known.

C-5(6) Sterol Desaturase from Tetrahymena thermophila: Gene Identification and Knockout, Sequence Analysis, and Comparison to Other C-5(6) Sterol Desaturases

ABSTRACT The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophilas enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.

Incomplete sterols and hopanoids pathways in ciliates: Gene loss and acquisition during evolution as a source of biosynthetic genes

Polycyclic triterpenoids, such as sterols and hopanoids, are essential components of plasmatic membrane in eukaryotic organisms. Although it is generally assumed that ciliates do not synthesize sterols, and many of them are indeed auxotrophic, a large set of annotated genomic sequences and experimental data from recently studied organisms indicate that they can carry putative genes and respond to the presence/absence of precursors in various ways. The pre-squalene pathway, for instance, is largely present in all sequenced ciliates except in Ichthyophthirius multifiliis; although Paramecium tetraurelia lacks the squalene synthase and Oxytricha trifallax the squalene hopene synthase, in addition to the former. On the other hand, the post-squalene pathway, requiring oxygen in several steps, is mostly incomplete in all ciliates analyzed. Nevertheless, a number of predicted genes, with high sequence similarity to C-4 methyl oxidase/s, C-14 demethylase, C-5 and C-7 desaturases and C-24 reductase of sterols are found in Tetrahymena and Paramecium, and scattered in other Stichotrichia ciliates. Moreover, several of these sequences are present in multiples paralogs, like the C-7 desaturase in Paramecium, that carries six versions of the only one present in Tetrahymena. The phylogenetic analyses suggest a mixed origin for the genes involved in the biosynthesis of sterols and surrogates in this phylum; while the genes encoding enzymes of the pre-squalene pathway are most likely of bacterial origin, those involved in the post-squalene pathway, including the processing of sterols obtained from the environment, may have been partially retained or acquired indistinctly from lower eukaryotes or prokaryotes. This particular combination of diverse gene/s acquisition patterns allows for survival in conditions of poor oxygen availability, in which tetrahymanol and other hopanoids may be advantageous, but also conditions of excess oxygen availability and abundant sterols, in which the latter are preferentially phagocyte, and/or transformed. Furthermore, the possibility that some of the genes involved in sterol metabolism may have another biological function in the most studied ciliate T. thermophila, was also explored.

The sterol-C7 desaturase from the ciliate Tetrahymena thermophila is a Rieske oxygenase which is highly conserved in animals

The ciliate Tetrahymena thermophila incorporates sterols from its environment that desaturates at positions C5(6), C7(8), and C22(23). Phytosterols are additionally modified by removal of the ethyl group at carbon 24 (C24). The enzymes involved are oxygen-, NAD(P)H-, and cytochrome b5 dependent, reason why they were classified as members of the hydroxylases/desaturases superfamily. The ciliates genome revealed the presence of seven putative sterol desaturases belonging to this family, two of which we have previously characterized as the C24-de-ethylase and C5(6)-desaturase. A Rieske oxygenase was also identified; this type of enzyme, with sterol C7(8)-desaturase activity, was observed only in animals, called Neverland in insects and DAF-36 in nematodes. They perform the conversion of cholesterol into 7-dehydrocholesterol, first step in the synthesis of the essential hormones ecdysteroids and dafachronic acids. By adapting an RNA interference-by-feeding protocol, we easily screened six of the eight genes described earlier, allowing the characterization of the Rieske-like oxygenase as the ciliates C7(8)-desaturase (Des7p). This characterization was confirmed by obtaining the corresponding knockout mutant, making Des7p the first nonanimal Rieske-sterol desaturase described. To our knowledge, this is the first time that the feeding-RNAi technique was successfully applied in T. thermophila, enabling to consider such methodology for future reverse genetics high-throughput screenings in this ciliate. Bioinformatics analyses revealed the presence of Des7p orthologs in other Oligohymenophorean ciliates and in nonanimal Opisthokonts, like the protists Salpingoeca rosetta and Capsaspora owczarzaki. A horizontal gene transfer event from a unicellular Opisthokont to an ancient phagotrophic Oligohymenophorean could explain the acquisition of the Rieske oxygenase by Tetrahymena.

Phylogenomic analysis of integral diiron membrane histidine motif-containing enzymes in ciliates provides insights into their function and evolutionary relationships.

The Integral Membrane Histidine Motif-containing Enzymes (IMHME) are a class of binuclear non-heme iron proteins widely distributed among prokaryotes and eukaryotes. They are characterized by a conserved tripartite motif consisting of eight to ten histidine residues. Their known function is the activation of the dioxygen moiety to serve as efficient catalysts for reactions of hydroxylation, desaturation or reduction. To date most studies on IMHME were carried out in metazoan, phototrophic or parasitic organisms, whereas genome-wide analysis in heterotrophic free living protozoa, such as the Ciliophora phylum, has not been undertaken. In the seven fully sequenced genomes available we retrieved 118 putative sequences of the IMHME type, albeit with large differences in number among the ciliates: 11 sequences in Euplotes octocarinatus, 7 in Ichthyophthirius multifiliis, 13 in Oxytricha trifallax, 18 in Stylonychia lemnae, 25 in Tetrahymena thermophila, 31 in Paramecium tetraurelia and 13 in Pseudocohnilembus persalinus. The pool of putative sequences was classified in 16 orthologous groups from which 11 were related to fatty acid desaturase (FAD) and 5 to the fatty acid hydroxylase (FAH) superfamilies. Noteworthy, a large diversity on the number and type of FAD / FAH proteins were found among the ciliates, a feature that, in principle, may be attributed to peculiarities of the evolutionary process, such as gene expansion and reduction, but also to horizontal gene transfer, as we demonstrate in this work. We identified twelve putative enzymatic activities, from which four were newly assigned activities: sphingolipid Δ4-desaturase, ω3/Δ15 fatty acid desaturase, a large group of alkane 1-monooxygenases, and acylamide-delta-3(E)-desaturase, although unequivocal allocation would require additional experiments. We also combined the phylogenetics analysis with lipids analysis, thereby allowing the detection of two enzymatic activities not previously reported: a C-5 sterol desaturase in P. tetraurelia and a delta-9 fatty acid desaturase in Cohnilembus reniformis. The analysis revealed a significant lower number of FADs sequences in the spirotrichea ciliates than in the oligohymenophorea, emphasizing the importance of fatty acids trophic transfer among aquatic organisms as a source of variation in metabolic activity, individual and population growth rates, and reproduction.

Introducing the concept of biocatalysis in the classroom: The conversion of cholesterol to provitamin D3

Biocatalysis is a fundamental concept in biotechnology. The topic integrates knowledge of several disciplines; therefore, it was included in the course “design and optimization of biological systems” which is offered in the biochemistry curricula. We selected the ciliate tetrahymena as an example of a eukaryotic system with potential for the biotransformation of sterol metabolites of industrial interest; in particular, we focused on the conversion of cholesterol to provitamin D3. The students work with wild type and recombinant strains and learn how sterol pathways could be modified to obtain diverse sterol moieties. During the course the students identify and measure the concentration of sterols. They also search for related genes by bioinformatic analysis. Additionally, the students compare biotransformation rates, growing the ciliate in plate and in a bioreactor. Finally, they use fluorescence microscopy to localize an enzyme involved in biotransformation. The last day each team makes an oral presentation, explaining the results obtained and responds to a series of key questions posed by the teachers, which determine the final mark. In our experience, this course enables undergraduate students to become acquainted with the principles of biocatalysis as well as with standard and modern techniques, through a simple and robust laboratory exercise, using a biological system for the conversion of valuable pharmaceutical moieties.