The ability of MALDI TOF MS (matrix-assisted laser desorption ionisation time-of-flight mass spectrometry) to identify cultivable microflora from two waste disposal sites from non-ferrous metal industry was analysed. Despite the harsh conditions (extreme pH values and heavy metal content in red mud disposal site from aluminium production or high heavy metal content in nickel sludge), relatively high numbers of bacteria were recovered. In both environments, the bacterial community was dominated by Gram-positive bacteria, especially by actinobacteria. High-quality MALDI TOF mass spectra were obtained but most of the bacteria isolates could be not identified using MALDI Biotyper software. The overall identification rate was lower than 20 %; in two of the environments tested identification rates were lower than 10 %. As a dominant bacterial species, Microbacterium spp. in drainage water from an aluminium red mud disposal site near Žiar nad Hronom, Bacillus spp. in red mud samples from the same site, and Arthrobacter spp. from nickel smelter sludge near Sereď were identified by a combination of the Biolog system and 16S rRNA sequence analysis. As the primary focus of the MALDI TOF MS-based methodology is directed towards medically important bacteria, reference database spectra expansion and refinement are needed to improve the ability of MALDI TOF MS to identify environmental bacteria, especially those from extreme environments.

A simple and specific method for direct detection of bovine mastitis pathogens (Streptococcus agalactiae (GBS), Staphylococcus aureus and Escherichia coli) in milk products, bacterial samples from milk and isolated bacterial DNA was developed. The method is based on polymerase chain reaction (PCR) using sequence-specific primers only for GBS and species-specific primers derived from 16S and 23S rRNA for all chosen species. The presence of the gene of surface immunogenic protein (Sip) in bovine GBS isolates, described previously only in human GBS isolates was confirmed. The GBS detection was performed with the sequence coding for surface immunogenic protein from GBS human isolates designated as Sip specific sequence (SSS); this sequence was selected for specific primer design. The sequence is unique for GBS and was designed from a consensus of all known sip genes. The specific identification was shown on a collection of 75 GBS bovine isolates from different localities in Slovakia. All isolates were positive to SSS, 16S and 23S rRNA sequence. The 16S and 23S rRNA PCR detection was also performed with S. aureus and E. coli isolates and specific PCR products were also detected. The detection limit of this assay for milk products was 6 CFU/microL (i.e. 6000 CFU/mL) for GBS and E. coli, and 16 CFU/microL for S. aureus. This rapid, sensitive and specific diagnostic method can be performed within hours and represents an innovative diagnostic tool for the detection of milk pathogens in dairy products.

The yeast cell division cycle gene CDC6 was isolated by complementation of a temperature-sensitive cdc6 mutant with a genomic library. The amino acid sequence of the 48 kDalton CDC6 gene product, as deduced from DNA sequence data, includes the three consensus peptide motifs involved in guanine nucleotide binding and GTPase activity, a target site for cAMP-dependent protein kinase and a carboxy-terminal domain related to metallothionein sequences. A plasmid-encoded CDC6-beta-galactosidase hybrid protein was located at the plasma membrane by indirect immunofluorescence. Disruption experiments indicate that the CDC6 gene product is essential for mitotic growth.

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Glycoside hydrolase family 77 (GH77) contains prokaryotic amylomaltases and plant‐disproportionating enzymes (both possessing the 4‐α‐glucanotransferase activity; EC 2.4.1.25). Together with GH13 and GH70, it forms the clan GH‐H, known as the α‐amylase family. Bioinformatics analysis revealed that the putative GH77 amylomaltase (MalQ) from the Lyme disease spirochaete Borrelia burgdorferi genome (BB0166) contains several amino acid substitutions in the positions that are important and conserved in all GH77 amylomaltases. The most important mutation concerned the functionally important arginine positioned two residues before the catalytic nucleophile that is replaced by lysine in B. burgdorferi MalQ. Similar remarkable substitutions were found in two other putative GH77 amylomaltases from related borreliae. In order to confirm the exclusive sequence features and to verify the eventual enzymatic activity, the malQ gene from B. burgdorferi was amplified using PCR. A c. 1.5‐kb amplified DNA fragment was sequenced, cloned and expressed in Escherichia coli, and the resulting recombinant protein was preliminarily characterized for its activity towards glucose (G1) and a series of malto‐oligosaccharides (G2–G7). This study confirmed that the remarkable substitution of the arginine really exists and the GH77 MalQ protein from B. burgdorferi is a functional amylomaltase because it is able to hydrolyse the malto‐oligosaccharides as well as to form their longer transglycosylation products.

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Endolysins as a class of antibacterial enzymes are expected to become a very useful tool for many purposes to control spreading of, e.g., multiresistant bacteria in different environments. Their antimicrobial properties could be broadened or altered by mutagenesis, domain swapping or gene shuffling. Therefore, the specific designing of endolysins to achieve their desired properties is challenging. This work is focused on the in silico analysis of protein domains presence in sequences of phage and prophage endolysins, followed by the study of variety of domain combinations in the individual endolysin types. The multiple sequence alignment of endolysin sequences revealed the recognition of sequence types with typical domain arrangement and conserved amino acids, divided according to the target substrate in bacterial cell walls. The five protein families of catalytic domains are specifically occurring in dependence of bacterial Gram-type. The presence, types and numbers of binding domains within endolysin sequences were also studied. The obtained results enable a more targeted design of endolysins with required antimicrobial properties.

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