Danuta Plochocka

Binding and Spreading of ParB on DNA Determine Its Biological Function in Pseudomonas aeruginosa

ParB protein of Pseudomonas aeruginosa belongs to a widely represented ParB family of chromosomally and plasmid-encoded partitioning type IA proteins. Ten putative parS sites are dispersed in the P. aeruginosa chromosome, with eight of them localizing in the oriC domain. After binding to parS, ParB spreads on the DNA, causing transcriptional silencing of nearby genes (A. A. Bartosik et al., J. Bacteriol. 186:6983-6998, 2004). We have studied ParB derivatives impaired in spreading either due to loss of DNA-binding ability or oligomerization. We defined specific determinants outside of the helix-turn-helix motif responsible for DNA binding. Analysis confirmed the localization of the main dimerization domain in the C terminus of ParB but also mapped another self-interactive domain in the N-terminal domain. Reverse genetics were used to introduce five parB alleles impaired in spreading into the P. aeruginosa chromosome. The single amino acid substitutions in ParB causing a defect in oligomerization but not in DNA binding caused a chromosome segregation defect, slowed the growth rate, and impaired motilities, similarly to the pleiotropic phenotype of parB-null mutants, indicating that the ability to spread is vital for ParB function in the cell. The toxicity of ParB overproduction in Pseudomonas spp. is not due to the spreading since several ParB derivatives defective in oligomerization were still toxic for P. aeruginosa when provided in excess.

Farnesyl diphosphate synthase activity affects ergosterol level and proliferation of yeast Saccharomyces cerevisae

The yeast farnesyl diphosphate synthase (FPPS) gene was engineered so as to construct allelic forms giving various activities of the enzyme. One of the substitutions was F96W in the chain length determination region. The other, K197, conserved within a consensus sequence found in the majority of FPP and GGPP synthases, was substituted by R, E and V. An intricate correlation has been found between the FPPS activity, the amount of ergosterol synthesized and cell growth of a mutant strain defective in FPPS. About 40% of wt FPPS activity was sufficient to support normal growth of the mutant. With further decline of FPPS activity (20 down to 3%) the amount of ergosterol remained unchanged at ∼0.16% (vs dry weight), whereas growth yield decreased and lag times increased. We postulate that, in addition to ergosterol initiating and maintaining growth of yeast cells, FPP and/or its derivatives participate in these processes.

Compound heterozygosity of two missense mutations in the NADH-cytochrome b5 reductase gene of a polish patient with type I recessive congenital methaemoglobinaemia

A case of type I methaemoglobinaemia observed in a Polish subject with compound heterozygosity for two mutations in the reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) gene is described. One is a novel mutation 647T→C which leads to substitution of isoleucine by threonine at position 215 (I215T). This maternal mutation was found in several family members. A previously known mutation, 757G→A, leads to the replacement of valine by methionine at position 252 (V252M). The latter mutation was found also in the father and one of the two brothers. The effects of these mutations were analysed on a model of the human b5R protein obtained by homology modelling. Although both amino acid substitutions are located in the NADH‐binding domain, the whole protein structure, especially the region between the flavin adenine dinucleotide and NADH‐binding domains, is disturbed. The structural changes in the I215T mutant are less prominent than those in the V252M mutant. We presume that the 647T→C mutation is a type I mutation, however, it has not been observed in the homozygous state.

The role of ERG20 gene (encoding yeast farnesyl diphosphate synthase) mutation in long dolichol formation. Molecular modeling of FPP synthase.

The yeast Saccharomyces cerevisiae strain LB332 bearing a mutation in the ERG20 gene encoding farnesyl diphosphate synthase (FPPS) synthesizes significantly longer dolichols than the wild type strain FL100 (14-31 and 14-19 isoprene units, respectively). The measurement of the short chain prenyl alcohols excreted into the medium shows that increased amounts of geraniol, dimethylallyl and isopentenyl alcohols but not farnesol are synthesized by the mutant strain. The wild type FPPS synthesizes farnesyl diphosphate (FPP) as the only product. The K197E substitution, as opposed to F112A/F113S in avian FPPS, does not change product specificity. Consequently, the possibility that mutated yeast FPPS synthesizes longer polyprenols is unlikely. This is supported by additional evidence such as in vitro analysis of the mutated FPPS products and molecular modeling. We suggest that formation of longer dolichols in vivo is the result of a change in the isopentenyl diphosphate/farnesyl diphosphate ratio caused by the erg20 mutation which in turn affects the activity of cis-prenyltransferase.

Several mutations including two novel mutations of the glucose-6-phosphate dehydrogenase gene in Polish G6PD deficient subjects with chronic nonspherocytic hemolytic anemia, acute hemolytic anemia, and favism

DNA sequencing revealed seven different glucose‐6‐phosphate dehydrogenase (G6PD) mutations in G6PD deficient subjects from 10 Polish families. Among them we found two novel mutations: 679C→T (G6PD Radlowo, class 2) and a 1006A→G (G6PD Torun, class 1). Variant G6PD Radlowo was characterized biochemically. Both novel mutations were analyzed using a model of the tertiary structure of the human enzyme. The main chain of G6PD Torun is different from the wild‐type G6PD. The remaining mutations identified by us in deficient Polish patients were: 542A→T (G6PD Malaga), 1160G→A (G6PD Beverly Hills), 1178G→A (G6PD Nashville), 1192G→A (G6PD Puerto Limon), and 1246G→A (G6PD Tokyo). Variant Tokyo was found in four families. In one of them favism was the first clinical sign of G6PD deficiency and chronic nonspherocytic hemolytic anemia (CNSHA) was diagnosed later. Variants G6PD Nashville and G6PD Puerto Limon were accompanied by the silent mutation 1311C→T of the G6PD gene. Hum Mutat 14:477–484, 1999.

The effects of statins on the mevalonic acid pathway in recombinant yeast strains expressing human HMG-CoA reductase.

BackgroundThe yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism.ResultsStatins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis.ConclusionStatins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects.

Investigating the effects of statins on cellular lipid metabolism using a yeast expression system.

In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids.

Different Behaviors In Vivo of Mutations in the β Hairpin Loop of the DNA Polymerases of the Closely Related Phages T4 and RB69

The T4 and RB69 DNA replicative polymerases are members of the B family and are highly similar. Both replicate DNA with high fidelity and employ the same mechanism that allows efficient switching of the primer terminus between the polymerase and exonuclease sites. Both polymerases have a beta hairpin loop (hereafter called the beta loop) in their exonuclease domains that plays an important role in active-site switching. The beta loop is involved in strand separation and is needed to stabilize partially strand-separated exonuclease complexes. In T4 DNA polymerase, modification of the beta-loop residue G255 to Ser confers a strong mutator phenotype in vivo due to a reduced ability to form editing complexes. Here, we describe the RB69 DNA polymerase mutant with the equivalent residue (G258) changed to Ser but showing only mild mutator activity in vivo. On the other hand, deletion of the tip of the RB69 beta loop confers a strong mutator phenotype in vivo. Based on detailed mutational spectral analyses, DNA binding activities, and coupled polymerase/exonuclease assays, we define the differences between the T4 and RB69 polymerases. We propose that their beta loops facilitate strand separation in both polymerases, while the residues that form the loop have low structural constraints.

Identification of C-terminal hydrophobic residues important for dimerization and all known functions of ParB of Pseudomonas aeruginosa

The ParB protein of Pseudomonas aeruginosa is important for growth, cell division, nucleoid segregation and different types of motility. To further understand its function we have demonstrated a vital role of the hydrophobic residues in the C terminus of ParBP.a.. By in silico modelling of the C-terminal domain (amino acids 242–290) the hydrophobic residues L282, V285 and I289 (but not L286) are engaged in leucine-zipper-like structure formation, whereas the charged residues R290 and Q266 are implicated in forming a salt bridge involved in protein stabilization. Five parB mutant alleles were constructed and their functionality was defined in vivo and in vitro. In agreement with model predictions, the substitution L286A had no effect on mutant protein activities. Two ParBs with single substitutions L282A or V285A and deletions of two or seven C-terminal amino acids were impaired in both dimerization and DNA binding and were not able to silence genes adjacent to parS, suggesting that dimerization through the C terminus is a prerequisite for spreading on DNA. The defect in dimerization also correlated with loss of ability to interact with partner protein ParA. Reverse genetics demonstrated that a parB mutant producing ParB lacking the two C-terminal amino acids as well as mutants producing ParB with single substitution L282A or V285A had defects similar to those of a parB null mutant. Thus so far all the properties of ParB seem to depend on dimerization.

Novel beta-spectrin mutations in hereditary spherocytosis associated with decreased levels of mRNA

Hereditary spherocytosis (HS) is one of the most frequent and heterogeneous inherited haemolytic anaemias. It is associated with abnormalities of several erythrocyte membrane proteins. We investigated relative mRNA quantification of red blood cell membrane protein genes using real‐time quantitative polymerase chain reaction (qPCR) in order to better characterize HS cases and to select genes to search for mutations in patients with spherocytosis. qPCR experiments indicated that the spectrin β gene (SPTB) could be involved in anaemia pathogenesis. DNA analysis of SPTB in the HS subjects with decreased SPTB mRNA levels revealed the presence of five previously undescribed mutations: R1756X, 781delT and IVS22nt‐4G>A, 1502insA and IVS20nt‐2A>G.