Isolation and characterization of bacteriophage PhiBP 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.

Morphological, physiological and molecular biological characterisation of isolates from first cases of Acanthamoeba keratitis in Slovakia.

Acanthamoeba keratitis (AK) is a relatively rare disease worldwide. Over the past 10 years, five cases of AK were reported in Slovakia. Four preserved Slovak strains and one strain from the Czech Republic isolated from corneal scrapes of patients with AK are characterised in this study. Genotype identification of isolates is based on sequences of the PCR amplimer GTSA.B1 amplified from 18S ribosomal DNA. A strain isolated from the first patient in 1999 was classified as a rare sequence type T15. This is just the second report in which genotype T15 has been associated with AK. The other three Slovak strains were identified as belonging to the most common genotype T4. The only strain originating from the Czech Republic was classified as sporadically appearing sequence type T3. All isolates were also studied for their temperature tolerance and growth characteristics. The cythopatic effect was tested in vitro on Vero cell cultures.

Assessment of environmental enterococci: bacterial antagonism, pathogenic capacity and antibiotic resistance.

The properties of 166 environmental strains belonging to the seven enterococcal species were studied. Enterococci originated mainly from surface- and waste-waters. They were screened for the presence of enterocins, virulence factors, and antibiotic resistance. The presence of different enterocin genes (entA, entB, entP, ent31, entL50AB) was frequently observed in our enterococcal isolates, 109 strains contained at least one enterocin gene. The distribution of enterocin genes varied according to the species, the genes were present mainly in E. hirae and E. faecium. By enterocin spot assay, 10 isolates inhibited the growth of Listeria strains. To evaluate the pathogenic ability of isolates, the distribution of selected virulence genes (cylA, gelE and esp) was investigated, eleven strains were positive in some of these genes, five of them belonged to E. faecalis. Regarding the antibiotic resistance of isolates, only two strains were multiresistant and two strains (E. hirae and E. casseliflavus) were resistant to vancomycin.

Evaluation of different PCR-based approaches for the identification and typing of environmental enterococci.

The aim of the work was the evaluation of different PCR-based methods to found an appropriate identification and typing strategy for environmental enterococci. Environmental enterococci were isolated mainly from surface- and waste-waters. Species identification was provided by combination of phenotypic (Micronaut System, Merlin) and molecular detection methods (fluorescent ITS-PCR, ddl-PCR, REP-PCR, AFLP). Very similar results were observed among molecular methods, however several discrepancies were recognized during comparison of molecular and biochemical identification. Seven enterococcal species (E. faecium, E. hirae, E. casseliflavus, E. mundtii, E. faecalis, E. durans and E. gallinarum) were identified within 166 environmental isolates. The results obtained in this work attest the importance of PCR-based methods for identification and typing of environmental enterococci. The fluorescent ITS-PCR (fITS-PCR) showed the best results in order to identify the enterococci strains, the method used the automated capillary electrophoresis to separate the PCR products in a very rapid and precise way. The AFLP method was suitable to identify and characterize the isolates, while the REP-PCR can be used for species identification.

An exodeoxyribonuclease from Streptomyces coelicolor: expression, purification and biochemical characterization.

Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene (exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3'-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA>ssDNA>3'-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3'-phosphomonoesterase only at 3'-dAMP as a substrate. The optimal temperature for its activity was 57 degrees C in Tris-HCl buffer at optimal pH=7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg(2+), Co(2+), Ca(2+)) and its activity was strongly inhibited in the presence of Zn(2+), Hg(2+), chelating agents or iodoacetate.

Transcriptional profiling of bacteriophage BFK20: coexpression interrogated by "guilt-by-association" algorithm.

Global gene expression profiling of bacteriophage BFK20 infecting the industrial L-lysine producer Brevibacterium flavum CCM 251 was performed using DNA microarray. The relative gene expressions were measured in fourteen time samples collected during phage development. Phage genes were classified as early, middle, late or unassigned based on complex expression patterns during infection. Temporal classification of BFK20 genes was in concordance with previous predictions. However, proposed late regulatory genes were reclassified and new functional assignments for ORF55 were strongly suggested. Furthermore, we consider possible functions of other genes and their products regarding coexpression pattern by using "guilt-by-association" algorithm. Microarray results were validated using real-time RT-PCR. The detailed description of phage BFK20 transcriptional profile can answer the basic questions of its life cycle and it also can help to prevent phage contamination during industrial fermentation. In addition, this work presents the first complete microarray time course study of gene expression utilizing loop design.

Complete nucleotide sequence and genome analysis of bacteriophage BFK20--a lytic phage of the industrial producer Brevibacterium flavum.

The entire double-stranded DNA genome of bacteriophage BFK20, a lytic phage of the Brevibacterium flavum CCM 251--industrial producer of L-lysine--was sequenced and analyzed. It consists of 42,968 base pairs with an overall molar G + C content of 56.2%. Fifty-five potential open reading frames were identified and annotated using various bioinformatics tools. Clusters of functionally related putative genes were defined (structural, lytic, replication and regulatory). To verify the annotation of structural proteins, they were resolved by 2D gel electrophoresis and were submitted to N-terminal amino acid sequencing. Structural proteins identified included the portal and major and minor tail proteins. Based on the overall genome sequence comparison, similarities with other known bacteriophage genomes include primarily bacteriophages from Mycobacterium spp. and some regions of Corynebacterium spp. genomes--possible prophages. Our results support the theory that phage genomes are mosaics with respect to each other.

An extracellular endodeoxyribonuclease from Streptomyces aureofaciens.

Several extracellular DNases were detected after cultivation of Streptomyces aureofaciens B96 under submerged conditions. These DNases are nutritionally regulated and high content of amino acid nitrogen in cultivation medium repress their production. By varying cultivation conditions, there remained only two extracellular nuclease activities. The major one, extracellular endodeoxyribonuclease SaD I, was purified to homogeneity by ammonium sulfate precipitation, adsorption on Spheron, chromatography on Superose-12P followed by FPLC on MonoQ and final purification on HiTrapQ. The molecular weight of the purified SaD I determined by SDS-PAGE was 31 kDa. The DNase hydrolyses endonucleolytically both double-stranded and single-stranded circular and linear DNA. It does not cleave RNA and does not exhibit phosphodiesterase nor phosphomonoesterase activity. It requires a divalent cation (Zn2+, Co2+, Mn2+, Mg2+) and its activity optimum is at neutral pH (pH 7.2). The optimal temperature for DNA cleavage was 40 degrees C. Activity was strongly inhibited in the presence of phosphate, Hg2+, chelating agents or iodoacetate, but it was stimulated by addition of dimethyl sulphoxide.

The Brevibacterium flavum sigma factor SigB has a role in the environmental stress response.

We have previously cloned a gene encoding a SigB, a principal-like sigma factor in Brevibacterium flavum, which was induced by several stress conditions. To clarify the in vivo function of this sigma factor, the sigB gene was disrupted by a homologous recombination, replacing the internal essential coding region in B. flavum chromosome by a kanamycin resistance marker gene. This mutation dramatically decreased vegetative growth rates of B. flavum. Studies of the effect of the sigB mutation on growth and viability of the cells under conditions of stress showed that the sigB mutant had increased susceptibility to acid, salt, alcohol, heat and cold stress. The plasmid-born wild-type sigB gene complemented the mutation. Based on the results, we propose that SigB has a role in vegetative growth and in response to various environmental stresses.