From sequence to function: Exploring biophysical properties of bacteriophage BFK20 lytic transglycosylase domain from the minor tail protein gp15.

Bacteriophages have evolved different mechanisms of infection and penetration of bacterial cell walls. In Siphoviridae-like viruses, the inner tail proteins have a pivotal role in these processes and often encode lytic protein domains which increase infection efficiency. A soluble lytic transglycosylase (SLT) domain was identified in the minor tail protein gp15 from the BFK20 bacteriophage.

Endolysin EN572-5 as an alternative to treat urinary tract infection caused by Streptococcus agalactiae.

Streptococcus agalactiae (Group B Streptococcus, GBS) is an opportunistic pathogen causing urinary tract infection (UTI). Endolysin EN572-5 was identified in prophage KMB-572-E of the human isolate Streptococcus agalactiae KMB-572. The entire EN572-5 gene was cloned into an expression vector and the corresponding recombinant protein EN572-5 was expressed in Escherichia coli in a soluble form, isolated by affinity chromatography, and characterized.

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.

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.

Characterization of Clinical and Carrier and Prophage Contribution to the Strain Variability.

(group B Streptococcus, GBS) represents a leading cause of invasive bacterial infections in newborns and is also responsible for diseases in older and immunocompromised adults. Prophages represent an important factor contributing to the genome plasticity and evolution of new strains. In the present study, prophage content was analyzed in human GBS isolates.

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.

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.

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.

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.

Characterization of a phiBP endolysin encoded by the Paenibacillus polymyxa CCM 7400 phage.

Endolysin (gp1.2) from the Paenibacillus polymyxa CCM 7400 temperate phage phiBP has a modular structure consisting of an N-terminal region with a catalytic glycosyl hydrolase 25 domain and a C-terminal cell wall-binding domain. The entire gene of this endolysin and fragments containing its catalytic and binding domains separately were cloned into expression vectors and the corresponding recombinant proteins were expressed in Escherichia coli and purified by affinity chromatography.

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.

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.

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.

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.

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 M(r) 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.

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.

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%.

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.