Pleiotropic effect of anionic phospholipids absence on mitochondrial morphology and cell wall integrity in strictly aerobic Kluyveromyces lactis yeasts.

Cardiolipin and phosphatidylglycerol are anionic phospholipids localized to the inner mitochondrial membrane. In this study, it is demonstrated by fluorescence and transmission electron microscopy that atp2.1pgs1Δ mutant mitochondria lacking anionic phospholipids contain fragmented and swollen mitochondria with a completely disorganized inner membrane. In the second part of this study, it was shown that the temperature sensitivity of the atp2.1pgs1Δ mutant was not suppressed by the osmotic stabilizer glucitol but by glucosamine, a precursor of chitin synthesis. The atp2.1pgs1Δ mutant was hypersensitive to Calcofluor White and caffeine, resistant to Zymolyase, but its sensitivity to caspofungin was the same as the strains with the standard PGS1 gene. The distribution of chitin in the mutant cell wall was impaired. The glucan level in the cell wall of the atp2.1pgs1Δ mutant was reduced by 4-8 %, but the level of chitin was almost double that in the wild-type strain. The cell wall of the atp2.1pgs1Δ mutant was about 20 % thinner than the wild type, but its morphology was not significantly altered.

Absence of anionic phospholipids in Kluyveromyces lactis cells is fatal without F1-catalysed ATP hydrolysis.

We have shown in previous research that the loss of phosphatidylglycerol and cardiolipin caused by disruption of the PGS1 gene is lethal for the petite-negative yeast Kluyveromyces lactis . This present study demonstrates the role and mechanism of atp2.1 in the suppression of pgs1 lethality in K. lactis cells. Phenotypic characterization has shown that a strain lacking the phosphatidylglycerolphosphate synthase (atp2.1pgs1Δ) possessed a markedly impaired respiratory chain, very low endogenous respiration, and uncoupled mitochondria. As a result the mutant strain was unable to generate a sufficient mitochondrial membrane potential via respiration. The atp2.1 suppressor mutation enabled an increase in the affinity of F(1)-ATPase for ATP in the hydrolytic reaction, resulting in the maintenance of sufficient membrane potential for the biogenesis of mitochondria and survival of cells lacking anionic phospholipid biosynthesis.

A real-time PCR diagnostic method for detection of Naegleria fowleri.

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence. Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.

First confirmation of Bordetella pertussis occurrence in Slovakia by using real-time PCR.

By application of the real-time PCR we manage to confirm the diagnosis and occurrence of a disease, which is caused by Bordetella pertussis-pertussis. Using this method we have proven the presence of DNA of Bordetella pertussis in the biological materials (nasopharyngeal swabs). The presence of IS481 genome sequence of Bordetella pertussis was confirmed. This method of detection of pathogens seems to be very rapid, simple, and specific. In the case of adequate technical laboratory equipment it may become very suitable and important supporter in explanation and confirmation of the occurrence of bacterial infections.

The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase in Kluyveromyces lactis is essential and assigned to chromosome I.

The phosphatidylglycerolphosphate synthase (CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase, EC 2.7.8.5) is an essential enzyme in biosynthesis of cardiolipin. In this work we report the isolation, heterological cloning, molecular characterization and physical mapping of the Saccharomyces cerevisiae PEL1/PGS1 homologue from Kluyveromyces lactis. The pel1 mutant strain of S. cerevisiae was used to isolate this homologue by screening a K. lactis genomic library. The novel cloned gene was named KlPGS1. Its coding region was found to consist of 1623 bp. The corresponding protein exhibits 55% amino acid identity to its S. cerevisiae counterpart. The presence of the mitochondrial presequence indicates its mitochondrial localization. Sporulation and ascus dissection of diploids heterozygous for single-copy disruption of KlPGS1 revealed that the KlPGS1 gene, is essential in K. lactis. Using a DIG-dUTP-labeled DNA probe-originated from the KlPGS1 gene and Southern hybridization of contour-clamped homogeneous electric field (CHEF)-separated K. lactis chromosomal DNA, the KlPGS1 gene was assigned to chromosome I. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the Accession No. AY176328.