Search

This study investigates the modification of commercial cellulose acetate microfiltration membranes by supercritical solvent impregnation with thymol to provide them with antibacterial properties. The impregnation process was conducted in a batch mode, and the effect of pressure and processing time on thymol loading was followed. The impact of the modification on the membrane’s microstructure was analyzed using scanning electron and ion-beam microscopy, and membranes’ functionality was tested in a cross-flow filtration system. The antibiofilm properties of the obtained materials were studied against Staphyloccocus aureus and Pseudomonas aeruginosa, while membranes’ blocking in contact with bacteria was examined for S. aureus and Escherichia coli. The results revealed a fast impregnation process with high thymol loadings achievable after just 0.5 h at 15 MPa and 20 MPa. The presence of 20% of thymol provided strong antibiofilm properties against the tested strains without affecting the membrane’s functionality. The study showed that these strong antibacterial properties could be implemented to the commercial membranes’ defined polymeric structure in a short and environmentally friendly process.

The aminoglycoside resistance genes sgm from Micromonospora zionensis and kgmB from Streptomyces tenebrarius were cloned into a yeast expression vector to test whether the encoded prokaryotic methylases can modify the 18S rRNA A-site and thus confer resistance to G-418. Despite the detectable presence of mRNAs in yeast cells, neither G-418-resistant yeast transformants nor positive western blot signals were obtained. Neither methylase was capable of methylating 40S subunits despite very high conservation of the antibiotic rRNA binding sites. However, the results provide novel insight into the action of Sgm by showing that it methylates the same site as KgmB, i.e. G1405 in 16S rRNA.

Transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase gene (pac) by cAMP receptor protein (CRP) and phenylacetic acid (PAA) was studied by using operon fusions with divergent reporter gene (lacZ, and phoA) constructs. A 150 bp DNA segment essential for the regulation of pac gene transcription by CRP and PAA was defined.

The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6x histidine tag with and without an enterokinase recognition producing proteins His(6)-EK-GrmA and His(6)-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.

The transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase (pac) gene was studied by modifying DNA sequences responsible for promoter activation by cyclic AMP receptor protein (CRP). The nucleotide sequence of the 5′-flanking region of the pac gene contains putative tandem CRP binding sites positioned at −69/−70 and at −111/−112 with respect to the transcriptional start site. Our results obtained with either point mutations or insertion or deletion mutants (each of which rotated the helix structure at the CRP binding site one-half turn) showed significant decrease of penicillin amidase (PA) activity, suggesting the CRP as a major activator. In this study, the evidence for the importance of spacing between tandem binding sites for CRP as well as for their location related to the promoter core sequence has been provided. Involvement of integration host factor (IHF) as an additional regulatory protein in the pac gene transcription regulation was also analyzed. It is shown that activation of the pac gene transcription is elevated by IHF.