Clara B. Nudel

Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene

The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic‐interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS‐polyacrylamide gels and hydrolyses long acyl chain p‐nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg‐yolk emulsions. The N‐terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two‐step secretion mechanism and require a lipase‐specific chaperone. The identification of an export signai sequence at the N‐terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two‐step translocation mechanism. However, no chaperone‐encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disutphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary‐structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site‐directed mutagenesis, resulting in production of an inactive extracellular lipase.

Isolation of yeast with killer activity and its breeding with an industrial baking strain by protoplast fusion

Summary“Wild” strains of Saccharomyces cerevisiae were isolated from dairy products, bakery goods, fresh fruit and vegetables, and tested for killer activity. Four isolates out of 238 strains possessed killer activity. The best of these was converted to the petite form and hybridized with an industrial strain of Saccharomyces cerevisiae by protoplast fusion. Thirty-eight out of 104 isolates had killer activity, and some of these had good dough-raising activity as well.

Effect of nitrogen sources on the regulation of extracellular lipase production in Acinetobacter calcoaceticus strains

SummaryExtracellular lipase production and lipA expression were studied in A.calcoaceticus strains in relation to the nitrogen sources. Addition of casamino-acids or tryptone improved 2–3 times lipase yield as compared to ammonium, yeast extract or proteose-peptone. Further increase in lipase yield and enzyme stability were achieved when ammonium was also added. Differences in yields were not related to lipA transcription. Proteolytic activity was detected in culture broths simultaneous with lipase degradation. Post- transcriptional processes including enzyme protection, inactivation and secretion must be considered as important factors affecting lipase production.

Blue-light dependent inhibition of twitching motility in Acinetobacter baylyi ADP1: Additive involvement of three BLUF domain-containing proteins

Twitching motility in Acinetobacter baylyi ADP1 is inhibited by moderate intensities of blue light in a temperature-dependent manner (maximally at 20 °C). We analysed the involvement of four predicted blue-light sensing using flavin (BLUF)-domain-containing proteins encoded in the genome of this strain in the twitching motility phenotype. All four genes were expressed both in light and in darkness. A phylogenetic tree showed that one BLUF domain, ACIAD2110, grouped separately from the other three (ACIAD1499, ACIAD2125 and ACIAD2129). Individual knockout mutants of the latter three, but not of ACIAD2110, fully abolished the light dependency of the twitching motility response. Quantitative analysis of transcript level of the three genes showed a decreased expression in the light, with dark/light ratios of 1.65±0.28, 1.79±0.21 and 2.69±0.39, for ACIAD2125, ACIAD2129 and ACIAD1499, respectively. Double and triple knockouts of ACIAD1499, ACIAD2125 and ACIAD2129 confirmed the same phenotype as the corresponding single knockouts. Complementation of all the single knockouts and the triple knockout mutants with any of the three BLUF-domain-encoding genes fully restored the inhibition of twitching motility by blue light that is observed in the wild-type strain. A. baylyi ADP1 therefore shows a high degree of redundancy in the genes that encode BLUF-containing photoreceptors. Moreover, all plasmid-complemented strains, expressing any of the BLUF proteins irrespective of the specific set of deleted photoreceptors, displayed increased light-dependent inhibition of twitching motility, as compared to the wild-type (P<0.001). We conclude that the three genes ACIAD1499, ACIAD2125 and ACIAD2129 are jointly required to inhibit twitching motility under moderate blue-light illumination.

Influence of the nitrogen source on growth and ethanol production byPichia stipitis NRRL Y-7124

SummaryGrowth and ethanol production byPichia stipitis in media containing different nitrogen sources were compared. Ammonium sulphate (plus vitamins and trace elements) increased ethanol productivity 57% and ethanol-biomass yield 113%, compared to yeast extract. Consumption of ammoniu, amino-acids and total nitrogen was determined. Two ammonium effects, low uptake in complex media and fermentation stimulation in defined media are discussed in terms of ammonium- ammonia futile cycling and maintenance energy.

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.

Isolation of a Δ7-cholesterol desaturase from Tetrahymena thermophila

Abstract Cell-free preparations of Tetrahymena thermophila catalyze the direct desaturation of cholesterol to Δ7-dehydrocholesterol (provitamin D3). The activity was isolated in the microsomal fraction from Tetrahymena homogenates. Δ7-Desaturase activity was stimulated fivefold by the addition of 6 mM ATP. Other cofactors assayed, including NAD, NADP, NADH or NADPH, had no significant effect. The activity was found in microsomes prepared from stationary-phase cultures of the ciliate, grown either with or without added cholesterol, thus indicating that it is constitutively expressed in T. thermophila 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.

Identification of virulence markers in clinically relevant strains of Acinetobacter genospecies.

Nine Acinetobacter strains from patients and hospital environment were analyzed for virulence markers, quorum sensing signal production, and the presence of luxI and luxR genes. The strains had several properties in common: growth in iron limited condition, biofilm formation, and no active protease secretion. Significantly higher catechol production was determined in patient isolates (P < 0.03), but other invasiveness markers, such as lipase secretion, amount of biofilm, cell motility, antibiotic resistance, and hemolysin production, showed large variability. Notably, all members of the so-called A. calcoaceticus-A. baumannii complex, regardless of whether the source was a patient or environmental, secreted mediumto long-chain N-acyl homoserine lactones (AHL) and showed blue light inhibition of cell motility. In these strains, a luxI homologue with a homoserine lactone synthase domain and a luxR putative regulator displaying the typical AHL binding domain were identified.