Immunodetection and N-terminal sequencing of DNA replication proteins of bacteriophage BFK20 - lytic phage of Brevibacterium flavum.

Phages are excellent models for studying the mechanism of DNA replication in prokaryotes. Identification of phage proteins involved in phage DNA replication is the first prerequisite for elucidation of the phage replication module. We focused on replication proteins gp41 (a putative helicase from SF2 superfamily), gp43 (a RepA-like protein), and gp44 (a putative DNA polymerase A) of phage BFK20 grown in Brevibacterium flavum. To identify them in the phage-host system, we prepared antibodies to these proteins which were cloned and expressed in Escherichia coli as his-tagged recombinant proteins. After purification to homogeneity the recombinant proteins served for raising specific polyclonal antibodies in mice. Using these antibodies in Western blot analysis the phage proteins gp41, gp43 and gp44 were detected during the phage growth cycle. The proteins gp41 and gp43, prepared from cell lysate by ammonium sulphate precipitation, were N-terminally sequenced and found to contain the sequences N-SVKPRELR-C and N-MLGSTML-C, respectively. This means that gp41 starts with serine but not with common methionine. We consider these findings an initial but important step towards more thorough characterization of replication proteins of phage BFK20.

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases.

OBJECTIVES: The determination of gene mutations is important for the diagnosis and prognosis of various gastrointestinal cancers. The aim of our study was to develop a new procedure for the analysis of KRAS gene mutation by application of the real-time PCR method. BACKGROUND: The detection process requires discriminate trace amount of mutant allele in a large excess of wild-type DNA in various samples. METHODS: The real-time PCR based technique using hybridization probes for five most frequently KRAS codon 12 mutations and WT specific peptide nucleic acid (PNA) was performed. Our multiplex detection system was tested in various DNA samples (tissue, bile, pancreatic juice) of patients with different diagnoses of gastrointestinal tract disease obtained by endoscopy and ERCP. RESULTS: We designed and optimized the real-time PCR conditions and tested various amount of PNA in PCR reaction to suppress amplification of the wild-type DNA. We determined the interassay variability of the melting temperatures and the results of mutation testing were confirmed by DNA sequencing with the 100 % accuracy. Incidence of searched mutations was 67.5 % in cohort of 40 patients; for KRASG12D it was in 44.4 %, KRASG12V in 22.2 %, KRASG12S in 14.8 %, KRASG12A in 14.8 % and KRASG12C in 3.8 %. The sensitivity of the assays is 1x10-5. CONCLUSIONS: Advantages of this technique are rapidity, accuracy and it is generally easy to perform. This method can be adapted for synchronic detection of multiple mutations and after readjustment by other type mutation of KRAS gene may serve as useful clinical tool for analyzing point mutations in various clinical samples (Tab. 3, Fig. 3, Ref. 42).

Assay design and optimization of mutant-enriched PCR based method for detection of K-ras gene mutations in pancreatic carcinoma.

The aim of our work was to develop a fast, reliable and sensitive PCR method to detect K-ras mutations in various clinical samples. There is a need for an unimpeachable method for early diagnosis and/or screening of pancreatic cancer (PC). We optimized and subsequently analyzed four methods based on mutant-enriched PCR for the sensitivity, cost and time expense. Using the selected optimal method we examined codon 12 K- ras mutations in a study population of 59 patients with upper GIT malignancies. Reliability of the genotyping was confirmed by sequencing. By using the best of our modified mutant-enriched PCR methods we achieved sensitivity of 1:1 x 10(5). Further studies are necessary to determine the optimal biological material sampling in PC.

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. METHODS AND RESULTS: We tested adsorption of the phage BFK 20 to the corynebacterial cell surface. We observed strong adsorption ranging from ca 79 to 93% on the cells of B. flavum ATCC strains, but only ca 76% for B. flavum CCM 251. Minor adsorption for Brevibacterium lactofermentum BLOB (ca 13%) and no adsorption for Corynebacterium glutamicum RM3 were determined. BFK 20 infection had no significant effect on growth and viability of C. glutamicum and B. lactofermentum, but significantly influenced growth and viability of B. flavum ATCC 21127, 21128 and 21474. Cell growth stopped in short time after infection but with no lysis. Brevibacterium flavum CCM 251 cell growth was arrested too and lysis occurred. The Southern hybridization confirmed the presence of significant amount of BFK 20 DNA in samples from B. flavum CCM 251 and B. flavum ATCC strains after BFK 20 infection. Only weak hybridization signal was detected for DNA from infected cells of B. lactofermentum BLOB and no signal for C. glutamicum RM3. CONCLUSIONS: Based on the above results we suggest presence of a mechanism leading to abortive infection in B. flavum ATCC 21127, 21128 and 21474. In B. lactofermentum BLOB and C. glutamicum RM3 the adsorption barrier is more likely. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the knowledge on defence mechanisms of corynebacteria against bacteriophages.

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 of Staphylococcus aureus and Streptococcus agalactiae obtained from clinical material. Among the 100 isolates of S. aureus two different RMS type II were detected. The first was expressed in isolates 32 and 33 (Sau32 I and Sau33 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 of S. agalactiae revealed only one RMS; it was detected in two isolates (no. 16 and 23; Sag16 I and Sag23 I). Restriction endonuclease expressed by these isolates cleaved DNA in sequence 5'-CTG CA/G-3' (PstI isoschizomer). In RMS-positive S. aureus and S. agalactiae isolates plasmid DNA capable of replication in Escherichia coli and Bacillus subtilis was also detected and isolated.

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.

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

Tetracycline-producing strains of Streptomyces aureofaciens expressed SauLPI restriction-modification (R-M) system, which recognized specific DNA sequence 5'-GCCGGC-3' (isoschizomer Nael). The activation of the second R-M system SauLPII (5'-GAGCTC-3', isoschizomer of XhoI), 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 in S. aureofaciens strains has been considered.

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 Streptomyces aureofaciens, we identified two sigma70-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.

An origin of DNA replication from streptomycete phage phi U1.

A DNA fragment from phage phi U1 containing an origin of DNA replication was identified. This fragment, designated ori, was able to support the maintenance in Streptomyces lividans of a plasmid lacking a functional Gram-positive ori. The sequence of the minimal ori fragment was determined and analyzed. The minimal fragment conferring replication origin function contained a number of direct and inverted repeats. The absence of an open reading frame in this ori fragment indicates that host factors alone were sufficient to initiate replication at ori.

Construction of promoter-probe shuttle vectors for Escherichia coli and corynebacteria on the basis of promoterless alpha-amylase gene.

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

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

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

Expression of human cystathionine beta-synthase in Escherichia coli: purification and characterization.

Cystathionine beta-synthase (CBS) purification from mammalian tissues is complicated by proteolysis and enzyme aggregation. To surmount these difficulties, we cloned human CBS cDNA in tandem with the beta-galactosidase sequence of the fusion vector, pAX5-, then expressed the fusion protein, beta-galactosidase/CBS, in transformed Escherichia coli cells. Proteolytic treatment of the ammonium sulfate fraction of bacterial lysates with endoproteinase Xa liberated CBS which could then be separated from its fusion partner by DEAE-cellulose chromatography. This nearly homogeneous enzyme preparation was purified 140-fold over the crude bacterial lysate with nearly 50% recovery, and its specific activity, 210 U/mg protein, was comparable to that purified from human liver. The purified enzyme contained pyridoxal 5'-phosphate and exhibited positive cooperativity toward S-adenosyl-L-methionine (Hill coefficient = 5.2; Kact = 34 microM). Km values of the cloned enzyme in the absence of AdoMet are 3.1 and 1.1 mM for serine and homocysteine, respectively. They are virtually identical to those from human hepatic CBS. A Soret absorbance band (lambda max = 428 nm) which shifted to 448 nm after reduction with sodium dithionite revealed the presence of heme in the enzyme. Expression of the fusion protein in E. coli with subsequent purification represents the first time this enzyme has been isolated in sufficient quantities for biophysical and biochemical investigation.

Transsulfuration depends on heme in addition to pyridoxal 5'-phosphate. Cystathionine beta-synthase is a heme protein.

The first committed step of transsulfuration is catalyzed by cystathionine beta-synthase (CBS), a known pyridoxal 5'-phosphate (PLP) enzyme. The inferred amino acid sequences of rat liver CBS and rat liver hemoprotein H-450 are identical. We now confirm the presence of heme b in rat and human liver CBS. Heme almost entirely accounts for the visible spectrum of CBS rather than PLP. Human CBS, expressed in Escherichia coli, acquires heme b from the host bacteria. delta-Aminolevulinate supplementation during bacterial growth increases both the heme saturation and the specific activity of the homogeneous enzyme more than 3-fold. 1 mol of the 63-kDa CBS subunit binds 1 mol of each (heme and PLP). The presence of heme is required for PLP binding, and the amount of PLP bound is limited by the heme content. Removal of PLP, but not heme, from CBS is reversible. These findings suggest that heme is functionally incorporated into CBS only during protein folding. This report describes the first instance of an enzyme that depends upon both heme and PLP for its function.

Characterization and sequence analysis of the F2 promoter from corynephage BFK20.

F2 promoter from corynephage BFK20 was isolated and characterized. It was functional in Escherichia coli and Corynebacterium glutamicum. Cloning of the F2 promoter into the pJUP05 promoter probe vector caused an increase of the neomycin phosphotransferase II specific activity. According to the Northern blot hybridization the nptII gene was expressed from the cloned F2 promoter. The apparent transcription start point in E. coli and C. glutamicum was determined. The -35 region of F2 promoter showed high similarity to that of E. coli promoter consensus sequence, but its -10 region was G+C rich and had no significant homology to that.