Gabriela Bukovska

Towards functional proteomics of minority component of honeybee royal jelly: the effect of post-translational modifications on the antimicrobial activity of apalbumin2.

This study illustrates multifunctionality of proteins of honeybee royal jelly (RJ) and how their neofunctionalization result from various PTMs of maternal proteins. Major proteins of RJ, designated as apalbumins belong to a protein family consisting of nine members with Mr of 49–87 kDa and they are accompanied by high number of minority homologs derived from maternal apalbumins. In spite of many data on diversity of apalbumins, the molecular study of their individual minority homologous is still missing.

Cloning and transcriptional characterization of two sigma factor genes, sigA and sigB, from Brevibacterium flavum.

Using a DNA fragment containing the principal sigma factor gene hrdB of Streptomycesaureofaciens, we identified two ς70 -like genes in a library of Brevibacterium flavum. Sequence analysis of the complete genes revealed two ORFs coding for gene products of 498 and 331 amino acid residues, which showed the greatest similarity to SigA and SigB sigma factors from Brevibacterium lactofermentum. We designated them similarly sigA and sigB. Transcription of B. flavum sigA and sigB has been investigated by S1-nuclease mapping by using RNA from different growth phases and after exposure to several stress conditions. Both genes are transcribed from a single promoter with transcription start points of 368 bp and 25 bp upstream from the proposed translation initiation codon of the sigA and sigB genes, respectively. Whereas sigA is transcribed almost constitutively during growth and after stress conditions, expression of sigB is significantly induced after several stress conditions, like acid stress, ethanol shock, and cold shock. Expression of both genes is significantly reduced after heat shock. Considering these transcriptional results, and also on the basis of the similarity to other principal sigma factor genes, sigA probably encodes the functional principal sigma factor, and sigB might have a function in stress response.

DNA microarrays — techniques and applications in microbial systems

Genome projects produce a huge amount of sequence information. As a result, the focus of genomics research is turning toward deduction of functional information about newly discovered genes. Thus structural genomics paves the way for a new discipline called functional genomics by providing the information required for microarray manufacture. Microarray technology is the result of automation and miniaturization in the detection of differential gene expression. By using this technology one can make a parallel analysis of RNA abundance and DNA homology for thousands of genes in a single experiment. Over the past several years, this unique technology has been used to explore hundreds transcriptional patterns and genome differences for a variety of microbial species. Applications of microarrays extend beyond the boundaries of basic biology into diagnostics, environmental monitoring, pharmacology, toxicology and biotechnology. We describe comprehensive nature of DNA microarray technology with emphasis on fabrication of DNA microarrays and application of this technology in biological environment with primary accent on microbial systems.

Identification of a bifunctional primase-polymerase domain of corynephage BFK20 replication protein gp43.

Replication protein gp43 is a gene product of orf43, from the genome of corynephage BFK20 and carries two different domains. The C-terminal part of gp43 is similar to F4-type helicases and the N-terminal part resembles the rare primase-polymerase (prim-pol) domain. We expressed the 372 amino acids of the gp43 N-terminus in the pET expression system as recombinant protein gp43N with His-Tag fusion on both the N- and C-termini. The protein gp43N was purified by immobilized cobalt or nickel ion affinity chromatography. Gel filtration chromatography on Superose 12 showed that the purified protein elutes at an apparent molecular weight of 80 kDa, suggesting that it may be a dimer. We detected primase and DNA polymerase activities in gp43N using a simple method based on the determination of inorganic pyrophosphate and we demonstrated these two activities by polyacrylamide and agarose gel electrophoresis. In both primase and polymerase reactions, gp43N used only deoxyribonucleotides. By using defined single-stranded oligonucleotides as templates, we found that the primase is not highly sequence specific and does not require a specific trinucleotide for initiation of primer synthesis. The prim-pol domain of gp43 is the first such domain of a phage protein studied as an individual heterologous protein.

Resistance of corynebacterial strains to infection and lysis by corynephage BFK 20

Aims:  Defence mechanisms of the corynebacterial strains against corynephage BFK 20, which causes lysis of Brevibacterium flavum CCM 251.

Characterization of a complex restriction-modification system detected in Staphylococcus aureus and Streptococcus agalactiae strains isolated from infections of domestic animals

Characterization of classic type II restriction-modification systems (RMS) (restriction endonucleases and modification methyltransferases) was carried out in isolates ofStaphylococcus aureus andStreptococcus agalactiae obtained from clinical material. Among the 100 isolates ofS. aureus two different RMS type II were detected. The first was expressed in isolates 32 and 33 (Sau32 I andSau33 I); the targeting sequence was determined as 5′-GGN CC-3′ (Sau96 I isoschizomer). The second was found in isolates no. 90, 93, 96*, and 98 (Sau90 I,Sau93 I,Sau96* I,Sau98 I) and enzymes recognized sequence 5′-CTY RAG-3′ (SmlI isoschizomer). Analysis of 40 isolates ofS. agalactiae revealed only one RMS; it was detected in two isolates (no. 16 and 23;Sag16 I andSag23 I). Restriction endonuclease expressed by these isolates cleaved DNA in sequence 5′-CTG CA/G-3′ (PstI isoschizomer). In RMS-positiveS. aureus andS. agalactiae isolates plasmid DNA capable of replication inEscherichia coli andBacillus subtilis was also detected and isolated.

Construction of promoter-probe shuttle vectors forEscherichia coli and corynebacteria on the basis of promoterless α-amylase gene

We constructed new promoter-probe vectors forE. coli and corynebacteria based on the promoterless α-amylase gene originating fromBacillus subtilis. Vectors pJUPAE1 and pJUPAE2 are suitable for isolation of transcriptionally active fragments from plasmids, phages or genomic DNA α-Amylase activity can be easily visually detected on agar plates containing a chromogenic substrate, or by direct measurement of α-amylase activity.

Construction and characterization of new corynebacterial plasmids carrying the α-amylase gene

Novel corynebacterial plasmids carrying α-amylase gene fromBacillus have been constructed. The level of α-amylase expression depends on the size of the vector. The highest expression levels were measured in brevibacteria harboring pA61 plasmid.

Connection between foreign DNA replication and induced expression of the restriction-modification system in Streptomyces aureofaciens.

Tetracycline-producing strains ofStreptomyces aureofaciens expressedSauLPI restriction-modification (R-M) system, which recognized specific DNA sequence 5′-GCCGGC-3′ (isoschizomerNaeI). The activation of the second R-M systemSauLPII (5′-GAGCTC-3′, isoschizomer ofXhoI), which was silent during the growth cycle, after a foreign DNA transfer into this strain was observed. This phenomenon was tentatively explained as a response of the cells against the exogenous DNA entering the cells. The involvement of a SOS-like response in induction of R-M system genes inS. aureofaciens strains has been considered.

Isolation and characterization of bacteriophage ΦBP from Paenibacillus polymyxa CCM 7400

A bacteriophage PhiBP infecting Paenibacillus polymyxa CCM 7400 was isolated from culture lysate. Electron microscopy of lysate samples revealed the presence of bacteriophage particles with polyhedral heads 56 nm in diameter and flexible noncontractile tails 144 nm in length. The profile of PhiBP structural proteins resembles that of other bacteriophages. The PhiBP genome consists of double-stranded DNA of 43-kbp size. Homology search of sequenced DNA fragments from EcoRI digest revealed regions with significant similarity to other known bacteriophage genes. Regions similar to phage terminase genes were identified within the 1.2-kbp fragment. Three lytic genes, two holin genes and one endolysin gene were identified within the 2.5-kbp fragment. We tested the isolates of P. polymyxa CCM 7400 for the presence of phage DNA on bacterial chromosome using PCR amplification with primers derived from proposed terminase and holin gene sequences. We confirmed the presence of PhiBP DNA on P. polymyxa chromosome by Southern hybridization. The bacteriophage PhiBP was capable of causing lysis of a P. polymyxaPhiBP lysogen despite the presence of the phage DNA on bacterial chromosome. Therefore, we concluded that PhiBP was a virulent mutant phage.