Pawel Staczek

GYRASE AND TOPO IV MODULATE CHROMOSOME DOMAIN SIZE IN VIVO

In bacteria, DNA supercoil movement is restricted to subchromosomal regions or ‘domains.’ To elucidate the nature of domain boundaries, we analysed reaction kinetics for γδ site‐specific resolution in six chromosomal intervals ranging in size from 14 to 90 kb. In stationary cultures of Salmonella typhimurium, resolution kinetics were rapid for both short and long intervals, suggesting that random stationary barriers occur with a 30% probability at approximately 80 kb intervals along DNA. To test the biochemical nature of domain barriers, a genetic screen was used to look for mutants with small domains. Rare temperature‐sensitive alleles of DNA gyrase and Topo IV (the two essential type II topoisomerases) had more supercoil barriers than wild‐type strains in all growth states. The most severe gyrase mutants were found to have twice as many barriers in growing cells as wild type throughout a 90 kb interval of the chromosome. We propose that knots and tangles in duplex DNA restrain supercoil diffusion in living bacteria.

Evaluation of a PCR melting profile method for intraspecies differentiation of Trichophyton rubrum and Trichophyton interdigitale

In order to identify the source of infections caused by dermatophytes, as well as the pathogen transmission pathway, there is a need to determine methods that allow detailed genetic differentiation of the strains within the dermatophyte genera. In this work, a PCR melting profile (PCR-MP) technique based on the ligation of adaptors and the difference in melting temperatures of DNA restriction fragments was used for the first time for intraspecies genotyping of dermatophytes. Clinical isolates and reference strains of dermatophytes isolated from skin, scalp, toenails and fingernails were used for this study. PCR-MP and random amplification of polymorphic DNA (RAPD) were used to type 11 isolates of Trichophyton rubrum, 40 isolates of Trichophyton interdigitale and 14 isolates of Microsporum canis. The results distinguished five types (containing one subtype) characteristic for T. rubrum and seven types characteristic for T. interdigitale using the PCR-MP technique. Analysis conducted using RAPD revealed five types for T. rubrum and four types for T. interdigitale isolates. No differentiation was observed for the M. canis isolates with either method. These results demonstrate that PCR-MP is a reliable method for the differentiation of T. rubrum and T. interdigitale strains and yields a discriminatory power that is at least equal to that of RAPD.

Proteus sp. – an opportunistic bacterial pathogen – classification, swarming growth, clinical significance and virulence factors

ABSTRACT The genus Proteus belongs to the Enterobacteriaceae family, where it is placed in the tribe Proteeae, together with the genera Morganella and Providencia. Currently, the genus Proteus consists of five species: P. mirabilis, P. vulgaris, P. penneri, P. hauseri and P. myxofaciens, as well as three unnamed Proteus genomospecies. The most defining characteristic of Proteus bacteria is a swarming phenomenon, a multicellular differentiation process of short rods to elongated swarmer cells. It allows population of bacteria to migrate on solid surface. Proteus bacteria inhabit the environment and are also present in the intestines of humans and animals. These microorganisms under favorable conditions cause a number of infections including urinary tract infections (UTIs), wound infections, meningitis in neonates or infants and rheumatoid arthritis. Therefore, Proteus is known as a bacterial opportunistic pathogen. It causes complicated UTIs with a higher frequency, compared to other uropathogens. Proteus infections are accompanied by a formation of urinary stones, containing struvite and carbonate apatite. The virulence of Proteus rods has been related to several factors including fimbriae, flagella, enzymes (urease - hydrolyzing urea to CO2 and NH3, proteases degrading antibodies, tissue matrix proteins and proteins of the complement system), iron acqusition systems and toxins: hemolysins, Proteus toxin agglutinin (Pta), as well as an endotoxin - lipopolysaccharide (LPS). Proteus rods form biofilm, particularly on the surface of urinary catheters, which can lead to serious consequences for patients. In this review we present factors involved in the regulation of swarming phenomenon, discuss the role of particular pathogenic features of Proteus spp., and characterize biofilm formation by these bacteria.

Development of transformation system for Trichophyton rubrum by electroporation of germinated conidia

Dermatophytes are the fungi that can cause infections of skin, hair, and nails due to their ability to utilize keratin. The genetic transformation systems of dermatophytes were successfully applied to Trichophyton mentagrophytes and Microsporum canis. Here we describe the procedure for genetic transformation of Trichophyton rubrum by electroporation of their germinated conidia. A linearized transformation vector (pCHSH75-Pch/GFP/TtrpC) containing bacterial hygromycin B phosphotransferase gene (hph) and green fluorescent protein gene (egfp) was introduced into the germinated conidia of T. rubrum by electroporation. PCR reaction analysis showed that egfp gene was integrated randomly and Southern blotting analysis demonstrated a single integration of hph gene into the chromosomal DNA of randomly selected transformant. In this work we report the efficient transformation and selection of the stable T. rubrum transformants.

Molecular properties prediction, docking studies, and antimicrobial screening of 1,3,4-thiadiazole and s-triazole derivatives.

A series of 1,3,4-thiadiazoles and s-triazoles were subjected to Molinspiration, ALOGPS 2.1, and Osiris programs to predict their molecular properties that are important for drug candidates. Subsequently, all of them were docked into the active sites of enzymes namely glucosamine-6-phosphate synthase (GlcN-6-P), VIM-2 metallo-β- lactamase (VIM-2), chitinase A1 (ChiA1), and sterol 14 alpha-demethylase (CYP51) that were considered in antimicrobial studies of thiadiazole and s-triazole derivatives. Since all compounds fulfilled the criteria for good membrane permeability, oral bioavailability, low toxicity and the potential inhibitory activities towards VIM-2, ChiA1, and CYP51, most of them were synthesized and their antimicrobial activity has been tested.