Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP.

The bacteriophage phiBP contains a newly discovered putative replisome organizer, a helicase loader, and a beta clamp, which together may serve to replicate its DNA. Bioinformatics analysis of the phiBP replisome organizer sequence showed that it belongs to a recently identified family of putative initiator proteins. We prepared and isolated a wild type-like recombinant protein, gpRO-HC, and a mutant protein gpRO-HCK8A, containing a lysine to alanine substitution at position 8. gpRO-HC had low ATPase activity regardless of the presence of DNA, while the ATPase activity of the mutant was significantly higher. gpRO-HC bound to both single- and double-stranded DNA substrates. Different methods showed that gpRO-HC forms higher oligomers containing about 12 subunits. This work provides the first information about another group of phage initiator proteins, which trigger DNA replication in phages infecting low GC Gram-positive bacteria.

A novel phage-encoded endolysin EN534-C active against clinical strain Streptococcus agalactiae GBS.

Streptococcus agalactiae (Group B Streptococcus, GBS) is primarily known as a major neonatal pathogen. In adults, these bacteria often colonize the gastrointestinal and urogenital tracts. Treatment of infections using antibiotics is often complicated by recurrences caused by multi-resistant streptococci. Endolysin EN534 from prophage A2 of human isolate Streptococcus agalactiae KMB-534 has a modular structure consisting of two terminal catalytic domains, amidase_3 and CHAP, and one central binding domain, LysM. The EN534 gene was cloned into an expression vector, and the corresponding recombinant protein EN534-C was expressed in Escherichia coli in a soluble form and isolated by affinity chromatography. The lytic activity of this endolysin was tested on cell wall substrates from different GBS serotypes, B. subtilis, L. jensenii, and E. coli. The enzyme lysed streptococci, but not beneficial vaginal lactobacilli. The isolated protein is stable in a temperature range of 20-37 °C. Calcium ions enhanced the activity of the enzyme in the pH range from 5.0 to 8.0. The exolytic activity of EN534-C was observed by time-lapse fluorescence microscopy on a S. agalactiae CCM 6187 substrate. Recombinant endolysin EN534-C may have the potential to become an antimicrobial agent for the treatment of S. agalactiae infections.

The helicase core accessory regions of the phage BFK20 DnaB-like helicase gp43 significantly affect its activity, oligomeric state and DNA binding properties.

The multifunctional phage replication protein gp43 is composed of an N-terminal prim-pol domain and a C-terminal domain similar to the SF4-type replicative helicases. We prepared four mutants all missing the prim-pol domain with the helicase core flanked by accessory N- and C-terminal regions truncated to varying extents. The shortest fragment still possessing strong ssDNA-dependent ATPase activity and helicase activity was gp43HEL519-983. The other proteins tested were gp43HEL557-983, gp43HEL519-855 and gp43HEL519-896. Removal of the 38 N-terminal residues in gp43HEL557-983, or the 128 and 87 C-terminal residues in gp43HEL519-855 and gp43HEL519-896, resulted in a significant decrease in the ATPase activities. The 38-amino acid N-terminal region has probably a function in modulating DNA binding and protein oligomerization. Deletion of the 87 C-terminal residues resulted in a twofold increase in the unwinding rate. This region is likely indispensable for binding to DNA substrates.

The BFK20 phage replication origin confers a phage-encoded resistance phenotype to the industrial strain Brevibacterium flavum.

The phage BFK20 replication origin was identified using bioinformatics tools and a fragment with the origin nucleotide sequence was cloned into the tetracycline resistance gene of Escherichia coli vector pBR328, to make the plasmid pBOS. After transformation into the host strain Brevibacterium flavum CCM 251, pBOS was able to replicate, showing that the cloned region may function as a replication origin. The presence of the BFK20 origin sequence in a pBOS plasmid isolated from B. flavum CCM 251 was confirmed by Southern hybridisation. Monitoring pBOS stability in corynebacterial hosts showed that pBOS was stable in Corynebacterium glutamicum RM3 for 20 generations and in B. flavum CCM 251 for 10 generations. The effect of the cloned BFK20 replication origin on host resistance to BFK20 infection was tested. Growth of a B. flavum CCM 251 strain harbouring pBOS stopped after phage infection, but without complete lysis. Five hours after infection, the viability of the modified strain was about five times higher than the viability of wild-type B. flavum CCM 251. Thus, the ability of the BFK20 replication origin to confer the origin-derived phage-encoded resistance phenotype to B. flavum CCM 251 was confirmed.

Identification of Brevibacterium flavum genes related to receptors involved in bacteriophage BFK20 adsorption.

Phage infection of bacterial cells is a process requiring the interaction between phage receptor binding proteins and receptors on the bacterial cell surface. We prepared a Brevibacterium flavum CCM 251 EZ-Tn5 transposon insertional library and isolated phage-resistant mutants. Analysis of the DNA fragments produced by single-primer PCR was used to determine the EZ-Tn5 transposon insertion sites in the genomes of phage-resistant B. flavum mutants. Seven disrupted genes were identified in forty B. flavum mutants. The phage resistance of these mutants was demonstrated by cultivation analysis in the presence of BFK20, and the adsorption rate of BFK20 to these mutants was tested. B. flavum mutants displayed significantly reduced adsorption rates; the lowest rate was observed for mutants containing interrupted major facilitator superfamily (MFS) protein and glycosyltransferase genes. Uninterrupted forms of these genes were cloned into corynebacterial vector pJUP06 and used for in trans complementation of the corresponding B. flavum mutants. The growth of these complemented mutants when infected with BFK20 closely resembled that of wild-type B. flavum. These complemented mutants also exhibited similar BFK20 adsorption as the wild-type control. We infer that the disrupted MFS protein and glycosyltransferase genes are responsible for the phage-resistant phenotype of these B. flavum transposition mutants.

Gp41, a superfamily SF2 helicase from bacteriophage BFK20.

Gp41 is one of two helicases encoded by the genome of bacteriophage BFK20. The gp41 sequence contains conserved motifs from the SF2 family of helicases. We prepared and studied three recombinant proteins: gp41HN, a wild type-like protein with an N-terminal His-Tag; gp41HC, with an S2A mutation and a C-terminal His-Tag; and gp41dC, a mutant protein with a deleted C-terminal region and His-Tags on both N- and C-termini. We tested the enzymatic activities and DNA binding abilities of these isolated proteins. We found that both gp41HN and gp41HC had strong DNA-dependent ATPase activities, but that the ATPase activity of gp41dC was significantly lower regardless of the presence of DNA. The preferred substrates for the NTP hydrolysis reactions were ATP and dATP. gp41HC and gp41HN exhibited a low helicase activity in a fluorescence-based assay using dsDNA substrates with a 3' overhang and with a forked end in the presence of ATP. We infer that the C-terminal region of gp41 may be involved in DNA binding, since removing this region in gp41dC reduced the protein's DNA binding ability.

Interaction between phage BFK20 helicase gp41 and its host Brevibacterium flavum primase DnaG.

Protein-protein interactions have been identified between the replication proteins of corynephage BFK20 and its host Brevibacterium flavum CCM 251. We tested the interactions between phage proteins gp40, gp41, gp42, gp43 and gp44 and between these phage proteins and host replication proteins DnaZX, DnaN, Dnaδ, DnaG, DnaA, RecF, TrxC, TrxB1 and SSB using a bacterial two-hybrid system. Phage proteins gp41, gp42 and gp43 self-associated, demonstrating that these proteins are oligomers in vivo. Interactions were also detected between phage protein gp41 and host proteins DnaZX, DnaN, Dnaδ, DnaG and SSB. ß-galactosidase activity measurements showed that the strongest interaction was between gp41 and DnaG. The interaction was studied further using 2-dimensional blue native SDS-polyacrylamide gel electrophoresis and Western blot analysis.

A RepA-like protein from bacteriophage BFK20 is a multifunctional protein with primase, polymerase, NTPase and helicase activities.

Phage BFK20 replication protein gp43 comprises an N-terminal prim-pol domain and a C-terminal domain similar to replicative helicases. We prepared and studied two recombinant proteins: gp43-1 containing both prim-pol and helicase domains, and gp43C with the helicase domain only. A SEC-MALS analysis indicated that gp43-1 forms a hexameric homooligomer. NTPase activity testing revealed that gp43-1 was able to hydrolyze a wide spectrum of NTPs, ATP the most efficiently. The ATPase activity of gp43-1 was strongly dependent on the presence of ssDNA in the reaction, but was low in the presence of dsDNA and in the absence of DNA. On the other hand, the ATPase activity of gp43C was very low regardless of the presence of DNA. The helicase activity of gp43-1 was detected using a fluorescence-based assay with a forked DNA substrate in the presence of ATP. However, no helicase activity could be detected for gp43C. We therefore conclude that the prim-pol domain is essential for the helicase and ssDNA-dependent ATPase activity of gp43-1.

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.

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.

Endolysin of bacteriophage BFK20: evidence of a catalytic and a cell wall binding domain.

A gene product of ORF24' was identified on the genome of corynephage BFK20 as a putative phage endolysin. The protein of endolysin BFK20 (gp24') has a modular structure consisting of an N-terminal amidase_2 domain (gp24CD) and a C-terminal cell wall binding domain (gp24BD). The C-terminal domain is unrelated to any of the known cell wall binding domains of phage endolysins. The whole endolysin gene and the sequences of its N-terminal and C-terminal domains were cloned; proteins were expressed in Escherichia coli and purified to homogeneity. The lytic activities of endolysin and its catalytic domain were demonstrated on corynebacteria and bacillus substrates. The binding activity of cell wall binding domain alone and in fusion with green fluorescent protein (gp24BD-GFP) were shown by specific binding assays to the cell surface of BFK20 host Brevibacterium flavum CCM 251 as well as those of other corynebacteria.

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.

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.